Relation between leaf and stem biochemical constituents and rooting ability of olive cuttings

Document Type: Research paper

Authors

1 Crop and Horticultural Science Research Department, Fars Agricultural and Natural Resources Research and Education Center, AREEO, Shiraz, Iran

2 Department of Horticultural Science, College of Agriculture, Shiraz University, Shiraz, Iran

3 Department of Horticultural Science , Darab College of Agriculture and Natural Resources, Shiraz University, Shiraz, Iran.

Abstract

The relationships between rooting potential with endogenous phenolic compounds, nitrogen and soluble carbohydrates of cuttings for five olive cultivars (‘Rowghani’, ‘Dusti’, ‘Tokhmekabki’, ‘Konservalia’ and ‘Amigdalolia’) were investigated. Extracts of the leaves and the base of the cuttings were used for analysis of biochemical compounds. Results showed that there were significant differences in rooting potential among olive cultivars. There was no correlation between leaves and stems nitrogen contents and rooting ability of the cuttings. Moreover, leaf and stem soluble sugars, leaf total phenolics, stem caffeic acid, narengin and chlorogenic acid contents of the leaf and stem, did not affect the rooting potential of the cuttings. However, leaf catechin, stem total phenolics and vanillic acid had positive effects on rooting potential of the cuttings. There was a negative correlation between rooting percentage of the cuttings and leaf gallic acid and narengine contents.

Keywords


Introduction

Olive (Olea europaea L.) is mainly propagated by leafy-stem cuttings. There is a variation in the rooting potential among olive cultivars and they have been classified into three groups namely: easy, moderate and hard-to-root cultivars (Denaxa et al., 2012). A balance among endogenous stimulatory and inhibitory factors, as well as, nutritional factors is required to promote rooting of olive cuttings (Wiesman and Lavee, 1995). Nitrogen levels appeared to be an important predictor for rooting potential of stem cuttings. In general, but not always, high N supply to the stock plants or high N content of cutting tissues decreases propagation success through cuttings (Porfírio et al., 2016; Dag et al., 2012; Hambrick et al., 1991; Druege et al., 2000). Nitrogen content strongly influences carbon allocation and partitioning in plants. Reduced propagation rate when high N levels are applied is due to reduction in starch reserves in the cutting tissue (Rowe et al., 1999). Carbohydrates are considered as optimal markers because they are the direct products of photosynthesis and constitute an important source of energy (Aslmoshtaghi and Shahsavar, 2010; Denaxa et al., 2012). Although many studies have shown a positive correlation between rooting potential and carbohydrate content of cuttings, others have failed to establish such correlation (Denaxa et al., 2012). It has been reported that seasonal changes in rooting ability of olive cuttings is related to the seasonal changes of carbohydrates in reproductive and vegetative shoots (Del-Rio et al., 1991). Cuttings of easy-to-root cultivars have been characterized with higher carbohydrate content than the cuttings of difficult-to-root cultivars (Denaxa et al., 2012; Yoo and Kim, 1996). Supply and redistribution of carbohydrates in cuttings may sometimes limit their rooting potential (Hartman et al., 2001).

Variations in rooting of cuttings have been associated with the changes in the levels of endogenous growth regulators and other metabolites. Such regulatory processes are controlled through qualitative and quantitative changes in enzymes, such as peroxidases, IAA-oxidase and polyphenol oxidase (Trobec et al., 2005).

Phenolic compounds play important roles in formation of adventitious roots (Aslmoshtaghi and Shahsavar, 2010). However, the mechanism by which phenolics act is still largely unknown. Phenolic compounds are secondary metabolites derived from pentose phosphate, shikimate and phenylpropanoid pathways in plants. They can be classified into non-soluble compounds such as tannins, lignins, cell-wal bound hydroxycinammic acid, and soluble compounds such as phenoic acids, flavonoids, phenylpropanoids and quinines. Phenolic compounds are the most widely occurring groups of phytochemicals in plants. They have considerable physiological and morphological importances and play a crucial role in plants growth and reproduction (Balasundram et al., 2006; Silva et al., 2006). Some phenolic compounds can affect adventitious root formation through preventing auxin degradation via IAA-oxidase system, forming auxin – phenol complex and inhibiting auxin decarboxylation (Haissig, 1986; Wilson and van Staden, 1990).

Several diphenols, o-diphenols, coumarins and polyphenols compounds can inhibit oxidation of IAA (Trobec et al., 2005). On the other hand, phenolic compounds such as monophenols and m-diphenols can inhibit the rooting process by stimulating IAA oxidation or promoting IAA decarboxylation, while some other phenolics have no regulatory effect on IAA content in plant tissues.

The objective of this study was to investigate the relationship between rooting potential of cuttings and different endogenous phenolic compounds, nitrogen contents and soluble carbohydrate in leaf and stem of five olive cultivars.

 

Materials and Methods

Plant materials, cutting preparation and propagation

This experiment was conducted in Agricultural and Natural Resources Research and Education Center of Fars province in June 2013 and June 2014. In spring (June) 15 cm long cuttings with 2-4 leaves and 0.8 cm diameter were taken from 25 years old mother plants of five olive cultivars namely: ‘Rowghani’ (easy-to-root), ‘Dusti’ and ‘Tokhmekabki’ (moderate-to-root), ‘Konservalia’ and ‘Amigdalolia’ (difficult-to-root). Rooting ability of cuttings were measured in two years (2013 and 2014), whereas the analysis of biochemical compounds were performed only in the second year (Jun, 2014). At the beginning of the experiment (only in 2014), the leaves and the basal 3 cm section of the cuttings were sampled for determination of their biochemical compounds. The basal ends of the cuttings were dipped in 4000 mg/l Indol-3-butyric acid (IBA) solution for 5 seconds. Thereafter, cuttings were planted in containers and placed on greenhouse benches that filled with a mixture of perlite and sand (1:1). The benches were bottom heated (22 ˚C) and the samples kept under an automatic mist and fogging unit (80% to 90% relative humidity). Each experimental unit consisted of 75 cuttings. At the end of the rooting period (3 months), the percentage of rooted cuttings were recorded. Cuttings with one or more clearly visible root initials (1-2 mm or longer) and/or roots were classified as rooted cuttings and were used for calculation of rooting percentage.

Extraction and analysis of phenolic compounds

Extraction, separation and quantification of phenolics were performed according to the method described by Misan et al. (2011) with some modifications.

Plant extracts were prepared by grinding 200 mg frozen samples with a solution of methanol/acetic acid (85:15) mixture for 24 h at 4 °C and were subsequently extracted in an ultrasonic bath at room temperature for 15 min. The resulting suspension was then centrifuged at 10000 rpm for 20 min at 0 °C. To remove compounds such as chlorophylls and lipids, the supernatant was extracted with 1 ml n-hexane and was centrifuged at 10000 rpm for 10 min. After removing the supernatant, the resulting solution was used for the analysis of total phenolic contents and their components.

Total phenolics were determined spectrophotometerically using Folin-Ciocalteu reagent and results were expressed as gallic acid equivalents. Quantification of total phenolics was performed using a microplate reader (Bio Tek ELx808) at 750 nm and gallic acid calibration curve (y= 119.02x – 22.348, R2= 0.9947).

Phenolic components were quantified by a HPLC (high performance liquid chromatograph) (Agilent 1200 series) equipped with a UV-Vis multi-wavelength detector at 280 and 330 nm. Data were evaluated using a Chemstation Software (Agilent Technologies) data processing system. The separation of components was achieved by an Agilent, XDB-C18, 5 μm, 4.6×150 mm column, at a flow-rate of 1 ml min-1. Solvent gradient was performed by varying the proportions of solvent A (methanol) to solvent B (2% acetic acid in water) for separation of galic acid, vanillic acid, catechin and naringin at 280 nm and chlorogenic acid and caffeic acid at 330 nm. The total running time and post-running time were 30 and 10 min, respectively. The column temperature was 30 ºC. The volumes of injected samples and standards were 20 μl which was done automatically using autosampler.

Nitrogen content

Samples were placed in paper bags and dried at 65 °C for 48 h in an oven. The dried samples were ground and passed from a 40 mesh screen, and stored for future analysis. To determine the total nitrogen, 0.3 g dried samples were used in a Kjeldahl instrument (KjelFlex K-360, Buchi Co., Switzerland) based on micro-Kjeldahl method (Bremner et al., 1996).

Soluble sugars

Samples were dried in a air-forced oven at 65 °C for 48 h and then ground into fine powder. Dried leaf and stem samples (100 mg) were used for measuring soluble sugars. Samples were extracted in hot 80% ethanol and assayed using phenol-sulfuric acid method using glucose as standard (Masuko et al., 2005).

Statistical analysis

The experiments were conducted based on a completely randomized design with three replications and 25 cuttings in each replication. One-way ANOVA was used for analysis of data by Excel and SAS programs. Means were compared using LSD test at α = 0.01 and 0.05.

Results

The means of rooting percentage for the five olive cultivars are shown in Figure 1. Significant differences were found among rooting percentages of the cuttings for the studied cultivars. Highest rooting percentage (31.3%) was observed in the easy-to-root cultivar ‘Rowghani’, while lowest rooting percentage, was observed in difficult-to-root cultivars ‘Konsrervalia’ (4.7) and ‘Amigdalolia’ (6.5%). Although, moderate-to-root cultivars (‘Dusti’ and ‘Tokhmekabki’) were ranked between easy-to-root and difficult-to-root cultivars, rooting percentage in ‘Tokhmekabki’ was significantly higher than rooting percentage in ‘Dusti’ (Fig. 1).

Fig. 1. Rooting percentage of cuttings for five olive cultivars. Columns followed by the same letters do not differ significantly at P≤0.05 using LSD test.

 

Although, no significant differences (P≤0.05) was found in stem nitrogen content of five olive cultivars (Fig. 2). There were considerable differences in leaf nitrogen content among the studied cultivars. The highest leaf nitrogen content (24.15 mg g-1 DW) was detected in ‘Tokhmekabki’ and the lowest leaf nitrogen content (19.15 mg g-1 DW) was found in ‘Amigdalolia’ cultivar. There was no significant relationship between the leaf nitrogen content and the rooting capacity among the cuttings of the five studied cultivars. Accordingly, no significant difference (P≤0.05) was found for leaf nitrogen content between ‘Tokhmekabki’ and ‘Konsrervalia’ cultivars with moderate and low rooting ability, respectively (Figs. 1 and 2).

Fig. 2. Leaf and stem nitrogen contents of five olive cultivars. Columns followed by the same capital or small letters do not differ significantly at P≤0.05 using LSD test.

 

The lowest content (54 mg g-1 DW) for leaf soluble sugars was detected in ‘Konservalia’; while highest content of leaf soluble sugars (69.2 mg g-1 DW) was found in ‘Rowghani’ cultivar. There was no statistical difference among other cultivars for leaf soluble sugars (Fig. 3).

Fig. 3. Leaf and stem total soluble sugars contents of five olive cultivars. Columns followed by the same capital or small letters do not differ significantly at P≤0.05 using LSD test.

 

A significant difference was found in the leaves of the cuttings with respect to their total phenol contents but these differences have no relation with rooting percentage and thus rooting potential of cuttings. Highest total phenol concentration in the stems (10.06 mg g-1 DW) was detected in ‘Tokhmekabki’ cuttings, while ‘Konservalia’ had the lowest phenol content (9.79 mg g-1 DW) among the studied cultivars (Fig. 4).

Fig. 4. Leaf and stem total phenol contents of five olive cultivars. Columns followed by the same capital or small letters do not differ significantly at P≤0.05 using LSD test.

 

Same amounts of caffeic acid, narengin and catechin were found in the stem cuttings of the cultivars. Highest and lowest leaf caffeic acid contents were found in ‘Rowghani’ and ‘Amigdalolia’ cuttings respectively, however, the leaf caffeic acid contents were not related to the rooting ability of the cuttings (Figs. 1 and 5). No differences were found in chlorogenic acid content in the leaf of the cuttings for the five studied cultivars (Fig. 6). Catechin was detected at relatively low concentrations (2.0 mg g-1 FW in ‘Tokhmekabki and 1.46 mg g-1 FW in ‘Rowghani’) and lower concentrations in the other cultivars (Fig. 11). A significant negative relationship was found between the rooting potential and leaf narengine concentrations among cultivar’s cuttings (Figs. 1 and 7). The same relationship was observed for galic acid contents in the leaves (Fig. 8). The highest concentration of vanillic acid was found in the leaves of ‘Rowghani’ cuttings, its concentrationwas slightly decreased in ‘Tokhmekabki’ and ‘Dusti’ and the lowest vanillic acid concentration was found in ‘Amigdalolia’ and ‘Konservalia’ cultivars (Fig. 9). Although C/N ratio in the leaf and stem of the cuttings was  statistically different among cultivars but have no clear pattern and therefore seems that it had no relation with rooting potential of the cuttings (Fig. 10). 

Fig. 5. Leaf and stem caffeic acid contents of five olive cultivars. Columns followed by the same capital or small letters do not differ significantly at P≤0.05 using LSD test.

Fig. 6. Leaf and stem chlorogenic acid contents of five olive cultivars. Columns Followed  by the same capital or small letters do not differ significantly at P≤0.05 using LSD test.

Fig. 7. Leaf and stem narengin contents of five olive cultivars. Columns followed by the same capital or small letters do not differ significantly at P≤0.05 using LSD test.

Fig. 8. Leaf and stem gallic acid contents of five olive cultivars. Columns followed by the same capital or small  letters do not differ significantly at P≤0.05 using LSD test.

Fig. 9. Leaf and stem vanillic acid contents of five olive cultivars. Columns followed by the same capital or small letters do not differ significantly at P≤0.05 using LSD test.

Fig. 10. Leaf and stem C/N ratio of five olive cultivars. Columns followed by the same capital or small letters do not differ significantly at P≤0.05 using LSD test.

Fig. 11. Leaf and stem catechin contents of five olive cultivars. Columns followed by the same capital or small  letters do not differ significantly at P≤0.05 using LSD test.

 

Results of regression analysis by stepwise method for leaf and stem variables and also their total (leaf and stem with together) are shown in Tables 1, 2 and 3 respectively. Data in Tables 1, 2 and 3 show intercept, probably values (Pr) and parameters estimated from these variables. Based on this method, variables entered into the model (step by step) and ultimately selected the variables having the most effect on the dependent variable (rooting percentage). The correlation coefficients (R2) of the equation were 0.99, 0.76 and 0.69 for leaf, stem and their total (leaf and stem with together), respectively.

Table 1. Stepwise regression analysis of leaf variables.

Parameters

Parameters estimated

Probably values

Intercept

43.17

0.011

Gallic acid

-0.089

0.0148

Vanillic acid

-2.106

0.0076

Narengin

0.715

0.0046

Caffeic acid

-3.94

0.0123

 

Table 2. Stepwise regression analysis of stem variables

Parameters

Parameters estimated

Probably values

Intercept

5.718

0.016

Gallic acid

-0.178

0.023

 

Table 3. Stepwise regression analysis of total (leaf+ stem) variables.

Parameters

Parameters estimated

Probably values

Intercept

2.834

<0.0001

Gallic acid

-0.078

0.0023

Carbohydrates

-0.023

0.0378

 

 

Discussion

The effects of tissue nitrogen contents on root initiation and development in stem cuttings depend on several factors such as carbohydrate availability, C/N ratio and interactions between endogenous hormones. It is believed that the function of nitrogen in root formation in cuttings is related to its role in the synthesis of nucleic acids and proteins. On the other hand, nitrogen redistribution in stem cuttings has been reported to be different among different cultivars during the rooting process (Hartman et al., 2001). Results of current study indicated that rooting capacity of olive cultivars was not related to nitrogen contents of their leaves or stems. Since, no differences were found among five olive cultivars for N content of their stem and no specified relation was found between changes in leaf N contents and rooting percentage of the cuttings (Figs. 1 and 2). The idea which rulled out the involvement of nitrogen contents in rooting ability can be further supported.Nitrogen is needed for the synthesis of different nitrogenous compounds, but the promotive effects of nitrogen in advantious root formation may also be considered when it has a close influence on carbohydrate contents, allocation, partitioning and metabolism (Porfírio et al., 2016; Dag et al., 2012; Hambrick et al., 1991; Druege et al., 2000; Scheible et al., 1997; Blazich, 1988).

It is well known that mobilization and translocation of carbohydrates in the olive stem cuttings generally have a positive effect on rooting ability of the cuttings (Aslmoshtaghi and Shahsavar, 2010; Denaxa et al., 2012). However, results obtained in the present study indicated that the rooting ability of the cuttings was not related to their total soluble sugar contents. These findings are in agreement with those reported by Tsipouridis et al. (2006). The absence of relationship between total sugares and rooting ability of the cuttings suggests that other biochemical, physiological and anatomical factors and/or the amounts of endogenous auxins and rooting co-factors might also be involved in adventious root formation in stem cuttings (Fouad et al. 1989; Denaxa et al. 2010; Denaxa et al., 2012; Del-Rio et al., 1991). Although many reserchers have reported that exogenous auxin application to the bases of the cuttings can increase carbohydrate and soluble sugare concentretion and hydrolizing enzyme on the cutting  sites,  the special role of soluble sugars in rooting has not been clarified yet (Ragonezi et al., 2010).

Although C/N ratio is important at the time of taking cutting from mother plants and their rooting ability, in this study the (soluble carbohydrate)/N ratio was not significantly related to rooting ability of the cuttings (Fig. 10). In agreement with our results, Hambrick et al. (1991) reported that in Rosa multiflora cuttings, starch/N and (total carbohydrate)/N ratios were more closley correlated with rooting than (soluble carbohydrates)/N.

Due to roles of carbohydrates as the source of energy and their requirements for macromolecules synthesis during the root formation process, some studies have focused on the changes of carbohydrates contents during rhizogenesis of woody cuttings (Denaxa et al., 2012; Murai et al., 1999; Pellicer et al., 2000; Welander, 1994). Hartman et al. (2001) described changes in carbohydrate levels during root initiation and development. They hypothesized that starch hydrolysis during rhizogenesis and release of soluble sugars in stem tissue can be used for root development. In our study, the time of cutting preparation (spring) did not seem to be a good time and does not coincide with the starch hydrolysis process in the studied olive cultivars. Usually, starch is hydrolyzed at the end of winter and the resulting sugars are consumed by vegetative growth whereas in the summer these soluble sugars are provided by photosynthesis. These findings are in agreement with the results of Denaxa et al. (2012). They found that the maximum rooting percentage of ʻArbequinaʼ was achieved in summer (76%) while the lowest rooting percentage was observed in spring (37%). In their study, the highest rooting percentage of the cuttings in summer coincided with the highest initial soluble sugars and the lowest starch concentrations in their tissues.     

The rooting potential of the five studied olive cultivars in our study have positive correlations with stem total phenol, catechin and vanillic acid concentrations but have negative correlation with leaf narengin and gallic acid contents (Figs. 7 and 8). Other phenolic compounds had no effects on the rhizogenesis of the cuttings for the studied cultivars. Previous studies have also shown that several phenolic compounds induce the effects of auxin on rooting by inhibiting the deactivation of IAA by IAA-oxidase (Faust, 1989; Haissig, 1986; Padney and Pathak, 1981; Wilson and van Staden, 1990) or by IAA decarboxylation (Wilson and van Staden, 1990). However, some of these compounds have inhibitory effects on root formation through inducing IAA oxidation or decarboxylation (De Klerk et al., 2011) or through acting as precursors for lignin formation (Haissig, 1986; Liu et al., 1996).

On the other hand, several phenolic compounds have no regulatory effects on the auxin content in plant tissues and as a result have no influence on root formation. Our study revealed that the individual phenolic compounds might have stimulatory or inhibitory effects on root formation. Fogaca and Fett-Neto (2005) investigated the role of auxin and its modulators in the adventitious root formation of two Eucalyptus species and found that tannic acid and phloroglucinal had no significant effects on the rooting response of the two Eucalyptus species.  Our study revealed that individual phenolic compounds might have stimulatory or inhibitory effects on root formation. Positively correlation was found between total phenol content of stem and rooting ability. This indicates that the balance between individual phenolic compounds and other endogenous compounds (such as auxins) is more important than their absolute concentrations alone. It has been reported that meta- and ortho- diphenols and polyphenols can inhibitauxin decarboxylation and as a result can promote rooting of cuttings but monophenols usually act as co-factors of IAA-oxidase and enhance IAA decarboxylation, consequently inhibit root formation (Bandurski et al., 1995; De Klerk et al., 2011).

In conclusion, it is hard to determine the involvement of total phenolic contents or individual phenolic compounds in root formation process of olive cuttings because they can fully mask the effect of each other. However, results of stepwise regression analysis suggested that leaf and stem gallic acid content in all cultivars had the highest negative correlation with the rooting ability of cuttings. Such negative correlations were found with lower intensities for leaf vanillic acid, narengin and caffeic acid contents. 

Aslmoshtaghi, E., and A.R. Shahsavar. 2010. Endogenous Soluble Sugars, Starch Contents and Phenolic Compounds in Easy and Difficult to Root Olive Cuttings. J. Biol. Environ. Sci. 4: 83-86.

Balasundram, N., K. Sundram, and S. Samman. 2006. Phenolic compounds in plants and agri-industrial by-products: Antioxidant activity, occurrence, and potential uses. Food Chem. 99(1): 191-203.

Bandurski, R.S., J.D. Cohen, J.P. Slovin, and D.M. Reinecke. 1995. Auxin Biosynthesis and Metabolism. In Plant Hormones. 39-65. Springer Netherlands.

Blazich, F.A. 1988. Mineral Nutrition and Adventitious Rooting. Adv. Plant Sci. Ser. 2: 61-69.

Bremner, J., D. Sparks, A. Page, P. Helmke, R. Loeppert, P. Soltanpour, M. Tabatabai, C. Johnston, and M. Sumner. 1996. Nitrogen-total. Methods of Soil Analysis Part 3-Chemical methods. 1085-1121.

Dag, A., R. Erel, A. Ben-Gal, I. Zipori, and U. Yermiyahu. 2012. The Effect of Olive tree Stock Plant Nutritional Status on Propagation Rates. Hort. Sci. 47(2): 307-310.

De Klerk, G.Jm, H. Guan, P. Huisman, and S. Marinova. 2011. Effects of Phenolic Compounds on Adventitious Root Formation and Oxidative Decarboxylation of Applied Indoleacetic acid in Malus ‘Jork 9’. Plant Growth Regul. 63: 175-185.

Del-Rio, C., L. Rallo, and J.M. Caballero. 1991. Effects of Carbohydrate Content on the Seasonal Rooting of Vegetative and Reproductive Cuttings of Olive. J. Hortic. Sci. 66: 301-309.

Denaxa, N.K., S.N. Vemmos, and P.A. Roussos. 2012. The Role of Endogenous Carbohydrates and Seasonal Variation in Rooting Ability of Cuttings of an Easy and a Hard to Root Olive Cultivars (Olea Europaea L.). Sci. Hort. 143: 19-28.

Denaxa, N.K., S.N. Vemmos, P.A. Roussos, and G. Kostelenos. 2010. The Effect of IBA, NAA and Carbohydrates on Rooting Capacity of Leafy Cuttings in three Olive Cultivars (Olea Europaea L.). In XXVIII International Horticultural Congress on Science and Horticulture for People (IHC2010): Olive Trends Symposium 924: 101-109.

Druege, U., S. Zerche, R. Kadner, and M. Ernst. 2000. Relation between Nitrogen Status, Carbohydrate Distribution and Subsequent Rooting of Chrysanthemum Cuttings as Affected by Pre-harvest Nitrogen Supply and Cold-storage. Ann. Bot. 85(5): 687-701.

Faust, M. 1989. Physiology of Temperate zone Fruit Trees. Wiley, New York, USA. P: 338.

Fouad, M.M., M.A. Fayek, H.H. Selim, and M.E. El-Sayed. 1989. Rooting of Eight Olive Cultivars under mist. In International Symposium on Olive Growing.286: 57-60.

Hambrick, C.E., F.T. Davies, and H.B. Pemberton. 1991. Seasonal Changes in Carbohydrate/nitrogen Levels During Field Rooting of Rosa multiflora ‘Brooks 56’ hardwood Cuttings. Sci. Hort. 46(1-2): 137-146.

Hartman, H.T., D.E. Kester, F.T. Davies, and R.L. Geneve. 2001. Plant Propagation Principles and Practices. Prentice-Hall, New Jersey. P: 656.

Haissig, B.E. 1986. Metabolic Processes in Adventitious Rooting of Cuttings. In New Root Formation in Plants and Cuttings. Springer Netherlands. 141-189.

Liu, Z.H., I.C. Hsiao, and Y.W. Pan. 1996. Effect of Naphthaleneacetic acid on Endogenous indole-3-acetic acid, Peroxidase and Auxin Oxidase in Hypocotyl Cuttings of Soybean During Root Formation. Bot. Bull. Acad. Sinica. 37: 247-253.

Masuko, T., A. Minami,  N. Iwasaki, and T. Majima. 2005. Carbohydrate Analysis by a Phenol-sulfuric acid Method in Microplate Format. Anal. Biochem. 339: 69-72.

Misan, A.C., N.M. Mimica-Dukic, A.I. Mandic, M.B. Sakac, I.L. Miloranovic, and I.J. Sedej. 2011. Development of a Rapid Resolution HPLC Method for the Separation and Determination of 17 Phenolic Compounds in Crude Plant Extracts. Cent. Eur. J. Chem. 9: 133-142.

Murai, Y., H. Harada, R. Mochioka, T. Ogata, S. Shiozaki, S. Horiuchi, T. Takagi. 1999. Relationships between rooting in softwood cuttings of mume (Prunus mume Sieb. et Zucc.) and sorbitol in shoots. J. Japan. Soc. Hort. Sci. 68: 648-654.

Padney, D., and R.K. Pathak. 1981. Effect of Rootstock, IBA and Phenolic Compounds on the Rooting of Apple Cuttings. Prog. Hort. 13: 105-110.

Pellicer, V., J.M. Guehl, F.A. Daudet, M. Cazet, L.M. Riviere, and P. Maillard. 2000. Carbon and Nitrogen Mobilisation in Larix Eurolepis Leafy Stem Cuttings Assessed by Dual C-13. N-15 labeling Relationships with Rooting. Tree Physiol. 20: 807-814.

Porfírio, S., M.D.G. Da Silva, M.J. Cabrita, P. Azadi, and A. Peixe. 2016. Reviewing Current Knowledge on Olive (Olea Europaea L.) Adventitious Root Formation. Sci. Hort. 198: 207-226.

Ragonezi, C., K. Klimaszewska, M.R. Castro, M. Lima, P. de Oliveira, and M.A. Zavattieri. 2010. Adventitious rooting of conifers: influence of physical and chemical factors. Trees. 24: 975-992.

Rowe, D.B., F.A. Blazich, and R.J. Weir. 1999. Mineral Nutrient and Carbohydrate Status of Loblolly Pine During mist Propagation as Influenced by Stock Plant Nitrogen fertility. Hort. Sci. 34(7): 1279-1285.

Scheible, W.R., A. Gonzalez-Fontes, M. Lauerer, B. Muller-Rober, M. Caboche, and M. Stitt. 1997. Nitrate Acts as a Signal to Induce Organic acid Metabolism and Repress Starch Metabolism in Tobacco. The Plant Cell. 9(5): 783-798.

Silva, S., L. Gomes, F. Leitao, A.V. Coelho, and L.V. Boas. 2006. Phenolic Compounds and Antioxidant activity of Olea europaea L. Fruits and Leaves. Food Sci. Technol Int. 12: 385-396.

Trobec, M., F. Stampar, R. Veberic, and G. Osterc. 2005. Fluctuations of Different Endogenous Phenolic Compounds and Cinnamic acid in the First Days of the Rooting Process of Cherry Rootstock ‘GiSelA 5’ leafy Cuttings. J. Plant Physiol. 162: 589-597.

Tsipouridis, C., T. Thomidis, and S. Bladenopoulou. 2006. Rhizogenesis of GF677, Early Crest, May Crest and Arm King Stem Cuttings During the Year in Relation to Carbohydrate and Natural Hormone Content. Sci. Hort. 108: 200-204.

Welander, M. 1994. Influence of Invironment, Fertilizers and Genotype on Shoot Morphology and Subsequent Rooting of Birch Cuttings. Tree Physiol. 15: 11-18.

Wiesman, Z., and S. Lavee. 1995. Enhancement of Stimulatory Effects on Rooting of Olive Cultivar Stem Cuttings. Sci. Hort. 62: 189-198.

Wilson, P.J., and J. van Staden. 1990. Rhizocaline, Rooting Co-factors, and the Concept of Promoters and Inhibitors of Adventitious Rooting- a Review. Ann. Bot. 66: 479-490.

Yoo, Y.K., and K.S. Kim. 1996. Seasonal Variation in Rooting Ability, Plant hormones, Carbohydrate, Nitrogen, Starch and Soluble Sugar Contents in Cuttings of White Forsythia (Abeliophyllum distichum Nakai.). J. Kor. Soc. Hortic. Sci. 37: 554-560.