Rooting of Aegiphila Brachiata Cuttings: Anatomical, Biochemical and Metabolomic Characterization

doi:10.21203/rs.3.rs-2957917/v1

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Rooting of Aegiphila Brachiata Cuttings: Anatomical, Biochemical and Metabolomic Characterization

https://doi.org/10.21203/rs.3.rs-2957917/v1

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To improve the production process of Aegiphila brachiata cuttings, the objective of this study was to evaluate the rhizogenic potential of cuttings from rejuvenated and adult mother plants collected in different seasons; we also characterized anatomically the cutting base, analyzed phenolic compounds and proteins contents, and identified polar metabolites. Two rooting experiments were carried out with cuttings from rejuvenated and adult mother plants, both collected in the four seasons of the year, submitted to treatments with indole butyric acid (0, 2000, 4000, 6000, and 8000 mg L^− 1 IBA), and kept in a greenhouse for 45 days. The experimental design was completely randomized in a double factorial scheme for cutting experiments (5 [IBA] x 4 seasons), and for biochemical analyses (2 mother plants x 4 seasons, material collected in the moment of rooting experiment installation and after 45 days); for metabolomic characterization, the 2 mother plants were evaluated only at summer experiment installation. Propagules from rejuvenated mother plants collected in the spring are more viable and indicated to produce A. brachiata cuttings (25.25% rooting), not requiring the use of IBA. Despite the higher rooting rate of cuttings from rejuvenated mother plants, there are no anatomical differences from adult mother plants cuttings. The highest contents of phenolic compounds were observed in propagules from rejuvenated mother plants in spring, indicating a positive effect of these compounds on the rhizogenic process. Maleic acid, 2-hydroxy-glutaric acid, mannose-6-phosphate, ribonic acid, glycine, and phosphoric acid were found in higher concentrations in cuttings from rejuvenated mother plants, with possible beneficial physiological effects on cuttings rooting.

Rhizogenesis. Vegetative propagation. Rejuvenation. Seasons. Native forest species. Metabolites

Aegiphila brachiata Vell. (Lamiaceae), popularly known as “peloteiro”, is a deciduous shrub-tree species native to the Atlantic Forest biome, occurring in the Southeast and South of Brazil (França 2003; Harley et al. 2015). The “peloteiro” is a plant with high biomass production and growth capacity in adverse conditions, important for the recovery of degraded ecosystems (França 2003), in addition to its fruits and leaves presenting food and medicinal potential, respectively. However, there are difficulties in producing seedlings of this high-potential species. Failure to germinate A. brachiata seeds is associated with a physical barrier imposed by the endocarp that prevents radicle protrusion (Maggioni et al. 2020). Based on this, vegetative propagation, mainly using the cutting technique, can be an excellent alternative for seedling production (Carpanezzi and Carpanezzi 2006; Oliveira and Ribeiro 2013), ensuring planting uniformity and higher productivity (Xavier et 2013; Wendling et al. 2016).

The process of root formation is complex, with metabolic changes occurring that include the action of enzymatic, hormonal, and other biochemical compounds acting as rooting inducers. The presence of biochemical compounds in adequate concentrations in the root formation zone can contribute to rhizogenesis (Lima et al. 2011; Porfírio et al. 2016). The central biochemical compounds involved in adventitious rooting are carbohydrates, which act as an energy resource (Ahkami et al. 2009); proteins, which help in the induction, formation, and development of adventitious roots and the signaling and biosynthesis of auxins (Husen 2008; Sairanen et al. 2012); and phenolic compounds, which are rooting cofactors by controlling IAA-oxidase activity (Shiozaki et al. 2013).

Another determining factor in the rhizogenic process is the age of the mother plant, given that propagules from plants in the juvenile growth phase have a greater rooting capacity than those from adult plants (Hartmann et al. 2018; Stuepp et al. 2018). The season of the year can also influence cuttings rooting, especially concerning the variation in endogenous hormonal balance throughout the seasons (Zuffellato-Ribas and Rodrigues 2001; Peña et al. 2015). Also, using plant regulators can accelerate the root formation process, increasing the rooting rate, the number and quality of roots, and rooting uniformity (Dias et al. 2012; Pires et al. 2013).

Through the anatomical characterization of propagules, it is possible to define the origin of adventitious roots and identify anatomical barriers to the emergence of root primordia. In general, propagules at a more advanced stage of development have anatomical characteristics, such as lignification and sclerification, which would be directly related to less rooting (Wendling et al. 2015).

Thus, this study aimed to evaluate the rhizogenic potential of cuttings from rejuvenated and adult mother plants of Aegiphila brachiata and the influence of different Indole-3-butyric Acid (IBA) concentrations and seasons of the year on the adventitious rooting of the species; we also analyzed the anatomy, the biochemical and the metabolomic profile of the cuttings, seeking to relate them to rhizogenesis. As far as we know, it is the first study to analyze the technical feasibility of producing seedlings of this species by cuttings and to address the importance of different metabolites in the rhizogenic process. Here we also bring relevant characterizations of A. brachiata, detecting compounds with pharmaceutical potential.

A. brachiata cutting technique

For the cutting of rejuvenated mother plants, herbaceous branches of A. brachiata were collected from a clonal garden in the field with specimens approximately two years old, originating from seedlings produced by stem cuttings, frequently pruned. And for the cuttings from adult mother plants, we collected semi-woody branches of 20 adult mother plants of natural occurrence in the understory of a Mixed Ombrophylous Forest. For both experiments, plants were in an area belonging to Embrapa Florestas in Colombo, Paraná, Brazil, and the collection of plant material took place in the four seasons of 2019.

Stem cuttings measuring 8 ± 1 cm in length were made from the plant material collected and disinfected with 0.5% sodium hypochlorite. After disinfestation, the bases of the cuttings were treated with different concentrations of indole butyric acid (IBA) in a hydroalcoholic solution (50% v/v) for 10 seconds: 0, 2000, 4000, 6000, and 8000 mg L^− 1. Planting was carried out in polypropylene tubes (53 cm³) filled with fine-grained vermiculite, and the cuttings were kept in a climate-controlled greenhouse with intermittent nebulization.

The experiment was established in a completely randomized design, in factorial scheme 5x 4, with five concentrations of IBA and four seasons of the year, with four replications of 20 cuttings per experimental unit. After 45 days of experiment installation, we evaluated rooting percentage, the number of roots per cuttings, length of the three largest roots per rooted cutting (cm), callogenesis percentage, leaf retention, shoots and mortality (cuttings with necrotic tissues).

Anatomical analyses

Cuttings’ bases of rejuvenated and adult mother plants were collected at the installation and during the evaluation of the cutting experiment in the summer. After fixation in FAA 70 (formaldehyde, acetic acid, and 70% ethanol) for 24 hours (Johansen 1940), the samples were stored in 70% ethanol.

The material was sectioned into 0.5 cm pieces and embedded in historesin (Leica®). The blocks were fixed in wooden tutors, and we obtained transverse cuts in a rotating microtome (Olympus® CUT 4055) with a thickness of 8 µm (Kraus and Arduin 1997). The sections were adhered to slides and stained with toluidine blue dye. Then, we analyzed and documented the slides using a Zeiss photomicroscope with a Sony® Cyber-shot P72 digital camera attached.

Biochemical analyses

Vegetative material and aqueous extract preparation

At the time of installation and evaluation (at 45 days) of each experiment in the four seasons of the year, 20 to 30 cuttings of rejuvenated and adult mother plants were randomly separated (composite sample), dried in microwaves for 5 minutes, crushed in an electric grinder and stored in a freezer (-20°C) until analyses.

To prepare the aqueous extracts, 20 mg of plant material was weighed in microtubes, and 2 mL of ethanol:water (1:1) was added. For extraction, the material was vortexed for 30 seconds, sustained in the Thermomixer® equipment for 1 hour at 60°C and 450 rpm, and next filtered. The extracts were prepared in triplicate for the determination of total phenolic compounds. Samples moisture was determined in triplicate by gravimetric method, drying in an oven at 105°C until constant mass. The results are expressed on a dry basis.

Total phenolic compounds and proteins

The content of total phenolic compounds was determined by Folin-Ciocalteu spectrophotometric method, as described by Singleton and Rossi (1965), with minor modifications. An aliquot of 0.15 mL of aqueous extract was added in a volumetric flask, followed by 0.5 mL of Folin-Ciocalteu reagent and 5 mL of distilled water. The solution was vortexed for 1 minute, and then 2 mL of 15% Na₂CO₃ aqueous solution was added. The solution was stirred for 30 seconds and adjusted to 10 mL with type 2 water. The solution was kept in dark for 2 hours, and the absorbance was determined in a spectrophotometer at 760 nm. The standard curve was obtained with gallic acid (3,4,5-trihydroxy benzoic acid) at concentrations from 0.25 to 10 mg L^-1, and the results expressed in mg equivalent to gallic acid (mg EAG g^-1 dry mass).

To analyze total proteins, 0.5 g of plant material was added into a digestion tube along with 0.5 g of catalyst (6.3 g of sodium selenite, 4.0 g of copper sulfate pentahydrate, and 48.5 g of anhydrous sodium sulfate). Then, 5.0 mL of concentrated sulfuric acid was added and kept in the digester block (Tecnal^®) at an initial temperature of 100ºC, raising the temperature by 50ºC every 30 minutes until reaching 350ºC, where it remained for a minimum of 3 hours. After complete digestion and cooling of the samples, 15 mL of distilled water was added. Sample distillation was performed in a nitrogen distiller (Tecnal^® TE036-1) and titration with hydrochloric acid (0.1M). Total protein concentration was determined according to the Kjeldahl method (AOAC, 1984) and expressed as mg g^-1 fresh mass.

Metabolomic analysis

For the metabolomic analysis, we evaluated rejuvenated and adult cuttings at the experiment installation. The determination of polar metabolites was performed according to Fiehn et al. (2000), with minor modifications.

For extraction, we weighed 20 mg of sample ground in liquid nitrogen, added 300 µL of pre-cooled methanol at -20°C, homogenized it in Vortex®, and kept it in a 2mL Eppendorf® in the freezer. Then, 30 µL of nonadecadonic acid (2 mg mL^− 1 stock in CHCl3 – used for qualitative internal control for the lipid phase) and 30 µL of ¹³C₆-sorbitol (used for quantitative internal standardization for the polar phase) were added. The sample was stirred for 15 min at 70°C and then cooled to room temperature. Afterwards, 200 µL of CHCl₃ was added, stirring for 5 min at 37°C. Next, 400 µL of water was added to the sample, homogenized in Vortex®, and centrifuged for 7 minutes at 12,000 rpm.

Two aliquots of the polar phase (top) were transferred to two 1.5 mL conical Eppendorff® tubes and dried in Speed Vac® for 16 hours without heating. Finally, a set of samples was derivatized in the sequence: methoxyamine hydrochloride in pyridine (50 µL, 20 mg mL^− 1, 40° C, 90 min) and MSTFA (N-methyl-N-(trimethylsilyl)-trifluoroacetamide, 50 µL, 40°C for 90 min) and analyzed by gas chromatography coupled to mass spectrometry (GC-MS, Focus, PolarisQ, Thermo Fisher®). 1 µL solution was injected by 1:25 flow division, and compounds were separated using a DB-5ms chromatographic column (30 m x 0.25 mm x 0.25 µm, Agilent®). The analysis was performed with constant flow and vacuum compensation with injector temperature at 230°C, transfer line at 250°C, and helium carrier gas at 1.0 mL min^− 1. The oven's internal temperature programming was 70°C with an isotherm of 1 min, heating at 320°C at a rate of 8°C min^− 1, and a final isotherm of 18 min. The mass spectrometer operation was in positive mode with electron ionization at 70 V and ion source temperature at 200°C.

In the AMDIS software (Automated Mass Spectral Deconvolution, NIST), the compounds were identified using mass spectra, a comparison of the retention index (IR), according to the Golm Metabolome Database platform (Hummel et al. 2010). Metabolite intensities (i.e., peak heights) were normalized by sample weight and internal standard (¹³C₆-Sorbitol), using fragments (m/z) characteristic of each compound.

Statistical analysis

For cutting technique, variances homogeneity were tested for by Bartlett's test (p < 0.05). Data were submitted to square root transformation for all variables that did not meet homogeneity assumptions, followed by an analysis of variance (p < 0.01 and p < 0.05); the variables that showed significant differences by the F test had their means compared by the Tukey test. We performed all analyses in the statistical software Assistat version 7.7.

For metabolites, data were analyzed using factor analysis, multivariate analysis of variance (MANOVA), and discriminant analysis (Marriott 1974; Kachigan 1991; Johnson and Wichern 1998; Anderson 2003; Hair Junior et al. 2005; Ferreira 2018). In the factor analysis, we considered that the factors should have an eigenvalue greater than 1 to be included. The extraction method used was principal components, with varimax rotation and variables standardization (correlation matrix).

MANOVA was carried out for the groups of variables obtained in the extracted factors, aiming to compare the propagules obtained from the different mother plants (rejuvenated and adult) using the average of the four seasons of the year. Pillai's Trace, Roy's Largest Root, Wilks' Lambda, and Hotteling's Trace tests were used to assess the significance of treatments. In case of significant differences between the types of material, the discriminant analysis was performed using the standardized variables and the mother plants of structures, canonical correlation, F test for each original variable, and the means of the treatments were evaluated.

Cutting of A. brachiata from rejuvenated mother plants

There was no rooting of cuttings in the autumn and winter seasons; for this reason, the statistical analysis of the rooting variables was based only on the summer and spring seasons and on the different IBA treatments (Table 1). Thus, according to ANOVA (p < 0.01 – Supplementary Table 1), there was no factor interaction for the variable rooted cuttings (R). The highest rooting percentage was obtained in spring (25.25%), differing statistically from summer (17.50%). No significant difference was observed between IBA concentrations at rooting.

Table 1

Percentage of cuttings rooting (R) from rejuvenated plants of *A. brachiata* collected in summer and spring, in different IBA treatments.
Source of variation	Variables
Seasons	R (%)
Summer	17.50	b
Spring	25.25	a
IBA (mg L^− 1)
0	18.12	a
2000	18.75	a
4000	19.37	a
6000	28.75	a
8000	21.87	a
CV(%)	52.52

Means followed by the same letter in the column do not differ statistically according to Tukey's test (p < 0.05).

Regarding the number and length of roots, there was an interaction of factors (p < 0.01) between seasons and IBA concentrations (Fig. 1). The highest value observed for both variables occurred in cuttings collected in spring with the application of IBA 4000 mg L^− 1.

For the other variables, the statistical analysis was based on the four seasons of the year, and there was no factor interaction (Supplementary Table 2). Regarding callogenesis percentage, there was a significant difference (p < 0.01) between the propagule collection seasons, with the highest value observed in cuttings collected during summer. The seasons also significantly influenced cutting mortality, with the highest value observed in winter (66.00%). Autumn was the season with the highest percentage of new sprouts on cuttings (30.50%), despite showing lower leaf maintenance (12.50%) (Table 2).

Table 2

Percentage of callogenesis (C), mortality (M), new shoots (NS), and leaf maintenance (LM) of cuttings from rejuvenated mother plants of *A. brachiata* in different seasons of the year and IBA concentrations.
Source of variation	Variables
Seasons	C (%)	M (%)	NS (%)	LM (%)
Summer	58.25 a	23.75 c	20.75 ab	53.25 a
Autumn	37.75 b	42.00 b	30.50 a	12.50 b
Winter	24.50 c	66.00 a	17.50 b	-
Spring	13.00 c	50.50 b	2.50 c	20.00 b
IBA
0 mg L ^− 1 IBA	30.00 ab	45.31 a	16.56 a	32.50 a
2000 mg L^− 1 IBA	28.12 b	52.50 a	17.81 a	27.50 a
4000 mg L^− 1 IBA	31.56 ab	50.62 a	15.00 a	21.67 a
6000 mg L^− 1 IBA	33.44 ab	39.69 a	19.37 a	32.50 a
8000 mg L^− 1 IBA	43.75 a	39.69 a	20.31 a	28.75 a

Means followed by the same letter in the column do not differ statistically according to Tukey's test (p < 0.05).

Cutting of A. brachiata from adult mother plants

According to ANOVA (p < 0.01), there was an interaction between seasons and IBA concentration for the percentage of rooting, callogenesis, and mortality (Supplementary Table 3). There was adventitious rooting only in the spring collection, and the IBA concentrations differed statistically in the rooting rates, with 4000 and 6000 mg L^− 1 IBA promoting greater rooting, despite the low rates (Table 3).

No differences were observed between IBA concentrations for the number and length of roots, and new shoots. As the cuttings only rooted in spring, the number of roots obtained in that season was 2.40, with an average length of 1.58 cm. Between seasons of collection, the spring showed the for the leaf maintenance variable, it was not possible to submit the data to statistical analysis due to the high number of null values.

Table 3

Percentage of rooting (R), callogenesis (C), and mortality (M) of cuttings from adult mother plants of *A. brachiata* in different seasons and IBA concentrations.
R (%)
Seasons		IBA (mg L^− 1)
Seasons		0	2000	4000	6000	8000
Spring		2.25 B	0.00 B	16.25 A	17.50 A	0.00 B
CV(%) 55.52
C (%)
Seasons		IBA (mg L^− 1)
Summer		5.00 abA	3.75 aA	11.25 aA	11.25 aA	12.50 aA
Autumn		0.00 bB	5.00 aA	0.00 aA	7.50 aA	0.00 bA
Winter		10.00 abA	11.25 aA	3.75 aA	6.25 aA	12.50 abA
Spring		18.75 aA	1.25 aB	10.00 aAB	6.25 aAB	0.00 bB
CV(%) 37.54
M (%)
Seasons		IBA (mg L^− 1)
Summer		92.50 aA	95.00 aA	88.75 aA	88.75 aA	87.50 abA
Autumn		63.75 bAB	56.25 bAB	65.00 bAB	51.25 bB	71.25 bA
Winter		90.00 aA	88.75 aA	96.25 aA	93.75 aA	87.50 abA
Spring		77.50 abB	97.50 aA	70.00 bB	76.25 aB	100.00 aA
CV(%) 50.17
NS (%)
Seasons			IBA (mg L^− 1)
Summer	2.45 bc			0	6.62 a
Autumn	16.30 a			2000	9.06 a
Winter	8.75 ab			4000	5.94 a
Spring	1.00 c			6000	6.56 a
				8000	7.44 a
CV(%) 128.67

Means followed by the same lowercase letter in the column and uppercase letter in the row do not differ statistically by Tukey's test (p < 0.05).

Anatomic analysis

In cuttings from rejuvenated and adult mother plants, we observed established secondary growth, characterized by the presence of secondary xylem and phloem and the beginning of periderm formation (Fig. 2a-b). In addition, the cortical region presented bundles of sclerenchyma fibers arranged in a discontinuous ring. The vascular cambium is very active, with a wide range of differentiating cells, with no apparent difference observed between the evaluated materials. The pith is composed of parenchyma cells, and the stem is covered by a uniseriate epidermis covered by a cuticle, with phellogen already active in the subepidermal layer (Fig. 2c-d). We verified that the root primordium formation occurred from the vascular cambium (Fig. 2f).

Biochemical composition

For total phenolic compounds (PC) and total proteins (TP) was a significant interaction between the type of mother plants and seasons of the year, both at the time of experiment installation (T0) and after 45 days in the rooting environment (T45). Analyzing the content of phenolic compounds at T0, the highest values for both rejuvenated and adult mother plants were obtained in spring, 37.39 and 25.81 mg g^− 1, respectively (Fig. 3a). In addition, we observed that the rejuvenated cuttings had a higher content of phenolic compounds or did not differ significantly from those from adult mother plants in all seasons and in both evaluations, except for winter in T45.

Regarding protein content, the highest value was obtained in cuttings collected in autumn, both for rejuvenated and adult mother plants, at T0 and T45 (Fig. 3b). The lowest levels in T0 were observed in cuttings collected in winter, for rejuvenated and adult plants. There was a reduction in the protein content during rooting for all treatments; that is, the values obtained for TP at T45 were lower than those obtained at T0, indicating a possible consumption of total proteins in the rooting process.

Metabolomic analysis

Regarding the polar metabolites, the analysis by GC-MS allowed the extraction of 59 compounds in the extracts of A. brachiata cuttings; however, of these, 14 are unknown metabolites - although they have already been found in other organisms, their chemical structure has not yet been elucidated. In this study, 45 compounds were identified, including amino acids (12), organic acids (8), sugars (8), acidic sugars (6), sugar alcohols (5), amines (2), phosphate (1), nitrogen compound (1), cyclitol (1), and phenolic compound (1) (Table 4).

Table 4

Metabolites identified in methanolic extract of cuttings of *A. brachiata.*
ID	RI	Compound	Class
C3	1097.9	Alanine	Aminoacids
C5	1209.6	Valine	Aminoacids
C8	1287.1	Isoleucine	Aminoacids
C10	1291.9	Proline	Aminoacids
C11	1299.7	Glycine	Aminoacids
C15	1353.7	Serine	Aminoacids
C17	1379.5	Threonine	Aminoacids
C20	1514.0	Aspartic acid	Aminoacids
C21	1522.8	cis − 4 - hydroxy – proline	Aminoacids
C27	1614.3	Glutamic acid	Aminoacids
C28	1620.7	Phentlalanine	Aminoacids
C34	1766.9	Glutamine	Aminoacids
C1	1053.1	Latic acid	Organic acids
C2	1076.6	Glycolic acid	Organic acids
C12	1300.5	Maleic acid	Organic acids
C13	1311.1	Succinic acid	Organic acids
C18	1481.5	Malic acid	Organic acids
C24	1569.5	2-hydroxy-glutaric acid	Organic acids
C37	1812.8	Citric acid	Organic acids
C39	1849.6	Quinic acid	Organic acids
C41	1871.7	Fructose	Sugar
C43	1911.0	Glucose	Sugar
C47	1969.3	Glucopyranose	Sugar
C54	2306.2	Mannose-6-phosphate	Sugar
C55	2624.9	Sucrose	Sugar
C57	2729.7	D-α,α-trehalose	Sugar
C59	2974.8	Galactinol	Sugar
C61	3347.0	Raffinose	Sugar
C14	1324.2	Glyceric acid	Acid sugars
C33	1753.6	Ribonic acid	Acid sugars
C45	1929.8	Glucuronic acid	Acid sugars
C48	1981.2	Galactonic acid	Acid sugars
C49	2001.8	Saccharic acid	Acid sugars
C51	2031.2	Galactaric acid	Acid sugars
C19	1500.2	Erythritol	Sugar alcohols
C29	1710.6	Arabitol	Sugar alcohols
C30	1715.2	Ribitol	Sugar alcohols
C44	1918.2	Mannitol	Sugar alcohols
C46	1926.6	Sorbitol	Sugar alcohols
C6	1260.2	Ethanolamine	Amines
C22	1524.5	4-amino-butanoic acid	Amines
C7	1266.0	Phosphoric acid	Phosphate
C16	1359.5	2 – piperidinecarboxylic acid	Nitrogen compound
C52	2081.2	Myo-inositol	Cyclitol
C60	3215.5	Luteoline	Phenolic compoud

ID: identification index; RI: retention index.

Through factor analysis, eight factors were extracted, which explain 94.7% of the total variance. From these factors, the first four were selected, explaining 78.51% of the total variance. The existence of a correlation of at least 0.60 was considered for the variable to be included in the factor. Once the analysis of variance (MANOVA) was performed for each of the groups of variables, a discriminant analysis was performed for groups 1 and 4, because only for these were differences between the types of mother plants (p < 0.001 and p < 0.05, respectively).

For the first factor, a discriminant function was determined, presenting a correlation between the variables evaluated and the discriminant function of 0.95. The discriminant function found selected the compounds maleic acid, 2-hydroxy-glutaric acid, mannose-6-phosphate, ribonic acid and glycine, as the variables of greatest weight. For the fourth factor, a correlation was observed between the variables evaluated and the discriminant function of 0.66. The discriminant function found selected only the phosphoric acid compound as the variable with the highest weight.

After the analyses of the averages of the original values, and the discriminating results, it was verified that propagules from rejuvenated mother plants have higher average contents of maleic acid, 2-hydroxy-glutaric acid, mannose-6-phosphate, ribonic acid, glycine, and phosphoric acid than those collected from adult breeders (Fig. 4).

The absence of rooting in the autumn and winter seasons are, for both propagules is possibly related to the period of vegetative rest of the species. Lower temperatures result in a decrease in plant metabolism, with lower vegetative growth and low auxin synthesis (Hartmann et al. 2018), which may interrupt rhizogenesis. In addition, variations in temperature and photoperiod throughout the year influence the endogenous levels of auxin, carbohydrates, and other chemical components present in parent plants (Costa et al. 2013; Pacurar et al. 2014).

The seasons also influence the degree of lignification of the propagules. Periods with lower vegetative growth cause a higher degree of lignification, resulting in lower rooting rates (Wendling et al. 2015; Zhang et al. 2016). Moreover, the cuttings collectes during winter had no leaves, because it is a deciduous species. The presence of leaves on the cuttings is of paramount importance in the rhizogenic process, as the leaves are sources of auxins, carbohydrates, and rooting cofactors, which are transported to the base of the cuttings, stimulating roots formation (Fragoso et al. 2015). Thus, the lack of rooting and the high mortality of cuttings in winter can be explained by the absence of leaves.

The highest rooting rates and number and length of roots were observed in spring. The greater number of roots results in higher absorption of nutrients and water by plants, and consequently, better seedlings’ survival and growth (Navroski et al. 2016; Duarte et al. 2019; Tsakaldimi et al. 2019). It is assumed that during spring, young leaves had a high photosynthetic capacity and, consequently, a high production of photoassimilates necessary for rhizogenesis. At the beginning of summer, the species began fruit filling, and part of these photoassimilates was translocated for reproductive development. Others studies also indicate spring as the most favorable season for rooting cuttings of other species (Ferriani et al. 2011; Nery et al. 2014; Sá et al. 2018); the higher temperatures affect plant metabolism, promoting faster chemical reactions and the higher auxin synthesis (Taiz et al. 2017). Thus, spring is the most suitable season for collecting species propagules because of greater adventitious rooting and root system development.

There was little influence of IBA on the rhizogenic process of cuttings from rejuvenated mother plants. Still, the rooting rates were not as promising as those obtained by Maggioni et al. (2020), with 53.75% rooting when using 6000 mg L^-1 IBA in cuttings collected from rejuvenated mother plants during spring, more than the double found in the present study (25.25%). When the endogenous level of auxin in the plant is insufficient, using more stable synthetic auxins, such as IBA, promotes a hormonal balance that favors root induction (Pires et al. 2013). Maggioni et al. (2020) observed an increase in rooting of cuttings as the concentration of IBA increased. It is known that the ideal concentration of these plant regulators varies according to the endogenous level of indole acetic acid (IAA) in each species (Erturk et al. 2010; Hartmann et al. 2018), in addition to factors such as matrice age (Stuepp et al. 2018), season of propagules collection (Oliveira and Ribeiro 2013), substrate (Ari 2016), among others. Thus, our results may have been influenced by other factors, resulting in lower rooting rates.

Contrary to what we observed with the rejuvenated material, the application of IBA favored the rooting of cuttings from adult plants. Thus, the differences between propagule maturation conditions may have influenced the rooting propensity. Propagules from plants in juvenile growth have physiological conditions that favor rooting, often not requiring the application of plant regulators (Dias et al., 2012; Wendling et al. 2014; Hartmann et al. 2018; Sá et al. 2018; Stuepp et al. 2018). While the adult material possibly had a lower concentration of endogenous auxin, depending on the exogenous application.

Leaf abscission occurred on a large scale in cuttings from adult mother plants. This process is related to the balance between the hormones auxin and ethylene. Low endogenous levels of auxins and high levels of ethylene lead to the formation of the abscission layer at the base of the leaf petiole, causing early leaf drop, as auxin acts as a suppressor of ethylene effects (Taiz et al. 2017). The high mortality rates observed in all treatments in the present study may be related to premature leaf fall, which is a source of photoassimilates necessary to supply the metabolic activities rhizogenesis. Thus, new methodologies could be tested with foliar application of auxins to delay leaves premature abscission and, in this way, contribute to cuttings survival and rooting.

Although the rooting of cuttings from rejuvenated mother plants was superior to that of adult cuttings, no anatomical differences were observed between the two materials. Several anatomical studies suggest a correlation between the difficulty in rooting cuttings and a continuous ring of sclerenchyma (Amissah et al. 2008; Lima et al. 2011; Porfírio et al. 2016), being a mechanical barrier in the emission of root primordia (Lima et al., 2011; Wendling et al. 2015). In A. brachiata, the discontinuity of the sclerenchymatous fibers ring probably does not make this tissue an impeding barrier to root emission, as it can be penetrated by the root primordium. We also did not notice a relationship between the presence of sclerenchymatous tissues and rhizogenic capacity since the two patterns of cuttings presented bundles of sclerenchymatous fibers and adventitious rooting occurred.

The origin of adventitious roots may come from the dedifferentiation of several cellular tissues, such as subepidermal tissues, cortex, cambium, secondary phloem, pericycle, or vascular bundles and varies from one species to another (Agulló-Antón et al. 2014; Denaxa et al. 2019). For A. brachiata, the root primordium was formed from the vascular cambium, evidenced by the proliferation of cells in this region, displacing adjacent structures to the periphery.

The highest concentrations of phenolic compounds were obtained in spring, the season where we observed the highest rooting rates for both rejuvenated and adult propagules; allowing us to assume that phenolic compounds positively influenced the rhizogenic process. In addition, generally, the rejuvenated cuttings had a higher content of phenolic compouds than cuttings from adult mother plants. Phenolic compounds are involved in the adventitious rooting of cuttings by influencing endogenous levels of IAA, which can stimulate or inhibit root formation (Schwambach et al. 2008; Shiozaki et al. 2013). Thus, it is suggested that the phenolic compounds may have influenced IAA levels, stimulating cuttings rooting.

There was a reduction in protein content during rooting. The values obtained for TP at T45 were lower than those obtained at T0, indicating a possible consumption of total proteins in the rooting process. Proteins are compounds with structural and enzymatic functions. Proteins with an enzymatic function are related to adventitious rooting, acting in cell division and differentiation (Husen 2008), signaling, and auxin biosynthesis, also acting in several other physiological processes (Sairanen et al. 2012). The highest protein levels were observed in autumn cuttings; some authors suggest that high protein levels in low-temperature seasons (autumn and winter), with reduced rooting rates, are due to the presence of proteins with structural functions, for example, proteins rich in glycine and proline (Bortolini et al. 2008; Ferriani et al., 2008). Other authors report a higher protein accumulation in the warmer seasons (spring and summer), suggesting that these proteins have an enzymatic function directly related to adventitious rooting (Sá et al. 2021). Our results did not show a relationship between protein accumulation and higher rooting rates; however, we point out that numerous other biochemical compounds may be involved in A. brachiata rooting, such as polar metabolites.

The metabolomics results suggest that evaluated polar metabolites had a beneficial effect on adventitious rooting, acting directly or indirectly in rhizogenesis. Organic acids, such as maleic acid and 2-hydroxy-glutaric acid, have multiple functions in plants in response to environmental stresses, such as pH regulation, osmotic adjustment, and detoxification (Kang et al. 2019; Andrade et al. 2021). Maleic acid is commonly found at low levels in plants due to its reduced involvement in biochemical pathways (Molero et al. 2019) and is described as a competitive inhibitor of several Krebs cycle enzymes (Evans and Garraway 1984). However, none of these acids are directly related to adventitious rooting according to literature. As for ribonic acid, there is no information about its role in plant physiology.

Accumulation of mannose-6-phosphate decreases the pool of pyrophosphate (Pi) required for ATP production (Herold and Lewis 1977) and represses transcription of genes associated with photosynthesis (Jang and Sheen 1997), potentially causing deleterious effects to plant growth and development. The presence of this compound would be negatively related to adventitious rooting by reducing the energy required for rhizogenesis; however, the highest levels of mannose-6-phosphate were observed in the propagules that showed the highest rooting capacity. Amino acids play an important role in adventitious root formation by promoting the synthesis of specific proteins necessary for root initiation and by being precursors of plant hormones (Taiz et al. 2017). Glycine is an abundant amino acid in chloroplasts and participates in the adjustment and protection of thylakoid membranes and thus helps in photosynthetic efficiency (Chen and Murata 2011). Thus, the accumulation of this compound in cuttings from rejuvenated mother plants may have benefited adventitious rooting by improving the production of assimilates necessary for rhizogenesis.

Phosphoric acid is an important compound in the supply of phosphorus to plants, which is an essential element incorporated into several organic compounds, such as sugars, phospholipids, nucleotides, and ATP (Taiz et al. 2017). The higher concentration of phosphoric acid in rejuvenated cuttings may have positively influenced the propensity for rooting, given the importance of phosphorus in plant metabolism, especially in energy storage.

In addition, it is noteworthy that eight sugars were identified in the extracts of A. brachiata cuttings. Sugars influence the emission and formation of roots because they are a source of carbon and energy for the biosynthesis of several substances essential to rhizogenesis. Sucrose can be directly used as an energy resource, assisting cell division to form the root primordium (Ahkami et al. 2009). Thus, higher sugar contents generally result in higher cuttings rooting percentages (Hartmann et al. 2011). Given the diversity of metabolites with distinct and vital functions in plant growth and development, further studies are necessary to evaluate the direct and indirect relationship between metabolites and root induction of propagules.

Furthermore, we observed the occurrence of compounds of pharmacological importance in the propagules of A. brachiata, such as luteolin and glutamine. Luteolin is a natural flavonoid with a high antioxidant capacity and anti-inflammatory effect, commonly found in species of the Lamiaceae family (Dhanokar et al. 2020; Picos-Salas et al. 2021). Glutamine is an amino acid of great importance to human health; there are reports of positive effects of glutamine supplementation in cancer patients, as it is an excellent immunomodulator (Fillmann et al. 2007; Urbina et al. 2017; Chitapanarux et al. 2020). Thus, we confirmed the synthesis of compounds with pharmaceutical relevance in the species, requiring in vitro and in vivo research to assess the medicinal potential of the “peloteiro”.

Propagules from rejuvenated mother plants is feasible and recommended to produce A. brachiata cuttings, not requiring the application of IBA for root induction. The seasons influence the species rhizogenic capacity, with spring being the best season for propagules collection. Despite the low rooting rate, there are no anatomical barriers that prevent species adventitious rooting, nor anatomical differences between cuttings from rejuvenated and adult plants.

The phenolic compounds seem to play a crucial role in the rhizogenesis of the species; while the total protein apparently does not influence the rooting process. In addition, 45 polar metabolites were identified in extracts of cuttings, predominantly amino acids. The metabolites maleic acid, 2-hydroxy-glutaric acid, mannose-6-phosphate, ribonic acid, glycine, and phosphoric acid were observed in higher concentrations in propagules from rejuvenated mother plants, possibly favoring the rhizogenic process.

Funding by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

Conflicts of interest/Competing interests The authors declare that they have no conflict of interest.

Acknowledgements: This work was partially supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

Agulló-Antón M, Fernández-Ayela A, Fernández-García N, Nicolás C, Albacete A, Pérez-Alfocea F, Sánchez-Bravo J, Pérez-Pérez JM, Acosta M (2014) Early steps of adventitious rooting: Morphology, hormonal profiling and carbohydrate turnover in carnation stem cuttings. Physiol Plant 150(3):446–462. https://doi.org/10.1111/ppl.12114
Ahkami AH, Lischewski S, Haensch K, Porfirova S, Horfmann J, Rolletschek H, Melzer M, Franken P, Hause B, Druege U, Hajirezaei MR (2009) Molecular physiology of adventitious root formation in Petunia hybrida cuttings: Involvement of wound response and primary metabolism. New Phytol 181(3):613–625. https://doi.org/10.1111/j.1469-8137.2008.02704.x
Amissah JN, Paolillo DJ JR, Bassuk N (2008) Adventitiouns root formation in stem cuttings of Quercus bicolor and Quercus macrocarpa and its relationship to stem anatomy. J Am Soc Hortic Sci 133(4):479–486. https://doi.org/10.21273/JASHS.133.4.479
Anderson TW (2003) An introduction to multivariate statistical analyses. J. Wiley, New York
Andrade A, Boero A, Escalante M, Llanes A, Arbona V, Gomez-Cadenas A, Alemano S (2021) Comparative hormonal and metabolic profile analyses based on mass spectrometry provides information on the regulation of water-deficit stress response of sunflower (Helianthus annuus L.) inbred lines with different water-deficit stress sensitivity. Plant Physiol Biochem 168:432–446. https://doi.org/10.1016/j.plaphy.2021.10.015
Ari E (2016) Effects of different substrates and IBA concentrations on adventitious rooting of native Vitex agnus-castus L. cuttings. Acta Sci Pol - Hortorum Cultus 15(2):27–41
Bortolini MF, Zuffellato-Ribas KC, Koehler HS, Carpanezzi AA, Deschamps C, Oliveira MC, Bona C, Mayer JLS (2008) Tibouchina sellowiana (Cham.) Cogn.: Enraizamento, anatomia e análises bioquímicas nas quatro estações do ano. Ciência Florestal 18(2):159–171. https://doi.org/10.5902/19805098454
Carpanezzi AA, Carpanezzi OTB (2006) Espécies nativas recomendadas para recuperação ambiental no Estado do Paraná, em solos não degradados. Comun Técnico - Embrapa 136
Chen THH, Murata N (2011) Glycinebetaine protects plants against abiotic stress: mechanisms and biotechnological applications. Plant Cell Environ 34(1):1–20. https://doi.org/10.1111/j.1365-3040.2010.02232.x
Chitapanarux I, Traisathit P, Chitapanarux T, Jiratrachu R, Chottaweesak P, Chakrabandhu S, Rasio W, Pisprasert V, Sripan P (2020) Arginine, glutamine, and fish oil supplementation in cancer patients treated with concurrent chemoradiotherapy: A randomized control study. CPCCR 44(1):1–10. https://doi.org/10.1016/j.currproblcancer.2019.05.005
Costa CT, Almeida MR, Ruedell CM, Schwambach J, Maraschin FS, Fett-Neto AG (2013) When stress and development go hand in hand: main hormonal controls of adventitious rooting in cuttings. Front Plant Sci 4:1–19. https://doi.org/10.3389/fpls.2013.00133
Denaxa NK, Vemmos SN, Roussos PA (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. https://doi.org/10.1016/j.scienta.2012.05.026
Dhanokar S, Kale M, Aher A, Gawali S, Patil R (2020) Luteolin- Phytoconstituents Responsible For Anti-Inflammatory Activity in Leaves of Vitex negundo Linn. (Lamiaceae). CTPPC 2(4):45–50. https://www.researchgate.net/publication/347459969
Dias CD, Oliveira LS, Xavier A, Wendling I (2012) Estaquia e miniestaquia de especiés florestais lenhosas do Brasil. Pesq Florest Bras 32(72):453–462. https://doi.org/10.4336/2012.pfb.32.72.453
Duarte MM, Mireski MC, Oliszeski A, Wendling I, Stuepp CA (2019) Rooting of yerba mate cuttings with different lengths. Rev UERGS 5(1):05–11. https://doi.org/10.21674/2448-0479.51.6-12
Erturk Y, Ercisli S, Haznedar A, Cakmakci R (2010) Effects of plant growth promoting Rhizobacteria (PGPR) on rooting and root growth of kiwifruit (Actinidia deliciosa) stem cuttings. Biol Res 43:91–98. https://doi.org/10.4067/S0716-97602010000100011
Evans RC, Garraway MO (1984) Effect of nitrogen-source and vitamins on ethanol and pyruvate production by bipolaris-maydis race. Mycopathol Mycol Appl 76:515–522. https://doi.org/10.1080/00275514.1984.12023872
Ferreira DF (2018) Estatística Multivariada. UFLA, Lavras
Ferriani AP, Lischka J, Mayer S et al (2008) Estaquia e anatomia de Vassourão-Branco. Sci Agrar 9:159–166. https://doi.org/10.5380/rsa.v9i2.10964
Ferriani AP, Zuffellato-Ribas KC, Helm CV, Boza A, Wendling I, Koehler HS (2011) Produção de brotações e enraizamento de miniestacas de Piptocarpha angustifolia. Pesq Florest Bras 31(67):257–264. https://doi.org/10.4336/2011.pfb.31.67.257
Fiehn O, Kopka J, Trethewey RN, Willmitzer L (2000) Identification of uncommon plant metabolites based on calculation of elemental compositions using gas chromatography and quadrupole mass spectrometry. Anal Chem 72(15):3573–3580. https://doi.org/10.1021/ac991142i
Fillmann H, Kretzmann NA, San-Miguel B, Llesuy S, Marroni N, González-Gallego J, Tuñón MJ (2007) Glutamine inhibits over-expression of pro-inflammatory genes and down-regulates the nuclear factor kappa B pathway in an experimental model of colitis in the rat. Toxicology 236(3):217–226. https://doi.org/10.1016/j.tox.2007.04.012
Fragoso RO, Witt NGPM, Obrzut VV, Valério S, Zuffellato-Ribas KC, Stuepp CA (2015) Maintenance of leaves and indolebutyric acid in rooting of juvenile Japanese Flowering Cherry cuttings. Rev Bras Ciênc Agrar 10(1):97–101. https://doi.org/10.5039/agraria.v10i1a5111
França F (2003) Revisão de Aegiphila Jacq. (Lamiaceae) e seu posicionamento sistemático. Thesis, Universidade de São Paulo
Hair Junior JF, Anderson RE, Tatham RL, Black WC (2005) Análise multivariada de dados. Bookman, Porto Alegre
Harley R, França F, Santos EP, Santos JS, Pastore JF (2015) Lamiaceae in Lista de Espécies da Flora do Brasil. Jardim Botânico do Rio de Janeiro. http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB8104
Hartmann HT, Davies FT, Geneve RL, Wilson SB (2018) Plant propagation: principles and practices. 9 th ed. Pearson, New York
Herold A, Lewis DH (1977) Mannose and green plants: occurrence, physiology and metabolism, and use as a tool to study the role of orthophosphate. New Phytol 79:1–40
Husen A (2008) Clonal propagation of Dalbergia sissoo Roxb. and associated metabolic changes during adventitious root primordium development. New Forest 36(1):13–27. https://doi.org/10.1007/s11056-007-9079-y
Jang JC, Sheen J (1997) Sugar sensing in higher plants. Trends Plant Sci 2:208–214. https://doi.org/10.1016/S1360-1385(97)89545-3
Johansen DA (1940) Plant microtechnique. McGraw-Hill, New york
Johnson RA, Wichern DW (1998) Applied multivariate statistical analyses. Prentice Hall, New Jersey
Kachigan SK (1991) Multivariate statistical analyses. Radius Press, New York
Kang ZY, Babar MA, Khan N, Guo J, Khan J, Islam S, Shrestha S, Shahi D (2019) Comparative metabolomic profiling in the roots and leaves in contrasting genotypes reveals complex mechanisms involved in post-anthesis drought tolerance in wheat. PLoS ONE 14(25):e0213502. https://doi.org/10.1371/journal.pone.0213502
Kraus JE, Arduin M (1997) Manual básico de métodos em morfologia vegetal. EDUR, Seropédica
Lima DM, Biasi LA, Zanette F, Zuffellato-Ribas KC, Bona C, Mayer JLS (2011) Capacidade de enraizamento de estacas de Maytenus muelleri Schwacke com a aplicação de ácido indol butírico relacionada aos aspectos anatômicos. Rev Bras Plantas Med 13(4):422–438. http://dx.doi.org/10.1590/S1516-05722011000400008
Maggioni RA, Vieira LM, Invernizzi SF, Carpanezzi AA, Zuffellato-Ribas KC (2020) Germination potential and vegetative propagation of Aegiphila brachiata Vell. Cerne 26(2):222–231. https://doi.org/10.1590/01047760202026022704
Marriott FHC (1974) The interpretation of multiple observations. Academic Press, London
Molero G, Tcherkez G, Roca R, Mauve C, Cabrera-Bosque L, Araus JL, Nogués S, Aranjuelo I (2019) Do metabolic changes underpin physiological responses to water limitation in alfalfa (Medicago sativa) plants during a regrowth period? Agric Water Manag 212:1–11. https://doi.org/10.1016/j.agwat.2018.08.021
Navroski MC, Nicoletti MF, Lovatel QC, Pereira MO, Tonett EL, Mazzo MV, Meneguzzi A, Felippe D (2016) Efeito do volume do tubete e doses de fertilizantes no crescimento inicial de mudas de Eucalyptus dunnii Maiden. Agrarian 9(31):26–33
Nery FSG, Zuffellato-Ribas KC, Koehler H (2014) Enraizamento de Psycotria nuda (Cham. & Schltdl.) Wawra (Rubiaceae) nas quatro estações do ano. Ciência Florestal 24(1): 243–250. https://doi.org/10.5902/1980509813341
Oliveira MC, Ribeiro JF (2013) Enraizamento de estacas de Euplassa inaequalis (Pohl) Engl. de mata de galeria em diferentes estações do ano. Biosci J 29:991–999
Pacurar DI, Perrone I, Bellini C (2014) Auxin is a central player in the hormone cross-talks that control adventitious rooting. Physiol Plant 151(1):83–96. https://doi.org/10.1111/ppl.12171
Peña MLP, Zanette F, Biasi LA (2015) Época de coleta e ácido indolbutirico no enraizamento de miniestacas de pitangueira. Semin Cienc Agrar 36(5):3055–3068. https://doi.org/10.5433/1679-0359.2015v36n5p3055
Picos-Salas MA, Heredia JB, Leyva-López N, Ambriz-Pérez DL, Gutiérrez-Grijalva EP (2021) Extraction Processes Affect the Composition and Bioavailability of Flavones from Lamiaceae Plants: A Comprehensive Review. Processes 9:1675–1713. https://doi.org/10.3390/pr9091675
Pires PP, Wendling I, Brondani G (2013) Ácido indolbutírico e ortotropismo na miniestaquia de Araucaria angustifolia. Revista Árvore 37(3):393–399. https://doi.org/10.1590/S0100-67622013000300002
Sá FP, Gomes EN, Maggioni RA, Wendling I, Helm CV, Sant’Anna-Santos BF, Zuffelatto-Ribas KC (2022) Biochemical and anatomical features of adventitious rhizogenesis in apical and basal minicuttings of Ilex paraguariensis. New Forest 53(5):411–430. https://doi.org/10.1007/s11056-021-09855-0
Sá FP (2018) Miniestaquia de erva-mate em quatro épocas do ano. Ciência Florestal 28(4):1431–1442. https://doi.org/10.5902/1980509835051. PortesD. C.Wendling, I.; Zuffellato-Ribas, K. C.
Sairanen I, Novák O, Pěnčík A, Ikeda Y, Jones B, Sandberg G, Ljung K (2012) Soluble Carbohydrates Regulate Auxin Biosynthesis via PIF Proteins in Arabidopsis. Plant Cell 24(12):4907–4916. https://doi.org/10.1105/tpc.112.104794
Schwambach J, Ruedell C, Almeida MR, Penchel R, Araújo E, Fett-Neto (2008) A. Adventitious rooting of Eucalyptus globulus x maidennii mini-cuttings derived from mini-stumps grown in sand bed and intermittent flooding trays: A comparative study. New Forest 36(3):261–271. https://doi.org/10.1007/s11056-008-9099-2
Shiozaki S, Makibuchi M, Ogata T (2013) Indole-3-acetic acid, polyamines, and phenols in hardwood cuttings of recalcitrant-to-root wild grapes native to east Asia: Vitis davidii and Vitis kiusiana. J Bot. http://dx.doi.org/10.1155/2013/819531
Singleton VL, Rossi JA Jr (1965) Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. AJEV 16(3):144–158
Stuepp CA, Wendling I, Xavier A, Zuffellato-Ribas KC (2018) Vegetative propagation and application of clonal forestry in Brazilian native tree species. Pesq agrop bras 53(9):985–1002. https://doi.org/10.1590/S0100-204X2018000900002
Tsakaldimi M, Ganatsas P, Jacobs DF (2013) Prediction of planted seedling survival of five Mediterranean species based on initial seedling morphology. New Forest 44(3):327–339. https://doi.org/10.1007/s11056-012-9339-3
Urbina JJO, San-Miguel B, Vidal-Casariego A, Crespo I, Sánchez DI, Mauriz JL, Culebras JM, González-Gallego J, Tuñón MJ (2017) Effects Of Oral Glutamine on Inflammatory and Autophagy Responses in Cancer Patients Treated With Abdominal Radiotherapy: A Pilot Randomized Trial. Int J Med Sci 14(11):1065–1071. https://doi.org/10.7150/ijms.20245
Wendling I, Brooks PR, Trueman SJ (2015) Topophysis in Corymbia torelliana × C. citriodora seedlings: adventitious rooting capacity, stem anatomy, and auxin and abscisic acid concentrations. New For 46(1):107–120. https://doi.org/10.1007/s11056-014-9451-7
Wendling I, Stuepp CA, Zuffellato-Ribas KC (2016) Araucaria clonal forestry: types of cuttings and mother tree sex in field survival and growth. Cerne 22(1):19–26. https://doi.org/10.1590/01047760201622012105
Wendling I, Trueman SJ, Xavier A (2014) Maturation and related aspects in clonal forestry – Part I: concepts, regulation and consequences of phase change. New Forest 45:449–471. http://dx.doi.org/10.1007/s11056-014-9421-0
Xavier A, Wendling I, Silva RL (2013) Silvicultura clonal: princípios e técnicas, 2 edn. Ed. da UFV, Viçosa
Zhang W, Fan J, Tan Q, Zhao M, Cao F (2016) Mechanisms underlying the regulation of root formation in Malus hupehensis stem cuttings by using exogenous hormones. J Plant Growth Regul 35:1–12. http://dx.doi.org/10.1007/s00344-016-9628-8
Zuffellato-Ribas KC, Rodrigues JD (2001) Estaquia: uma abordagem dos principais aspectos fisiológicos. [K. C. Zuffellato-Ribas], Curitiba

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Rooting of Aegiphila Brachiata Cuttings: Anatomical, Biochemical and Metabolomic Characterization

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INTRODUCTION

MATERIAL E METHODS

Anatomical analyses

Biochemical analyses

Vegetative material and aqueous extract preparation

Total phenolic compounds and proteins

Metabolomic analysis

Statistical analysis

RESULTS

Anatomic analysis

Biochemical composition

Metabolomic analysis

DISCUSSION

CONCLUSIONS

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