MORPHO-AGRONOMIC CHARACTERIZATION OF Varronia curassavica GERMPLASM CONSERVATED “EX SITU” CARACTERIZAÇÃO MORFOAGRONÔMICA DE GERMOPLASMA DE Varronia curassavica CONSERVADO “EX SITU”

Varronia curassavica Jacq. is a medicinal and aromatic plant native to Brazil. The essential oil of this species is valued by the pharmaceutical industry due to its bioactive substances containing anti-inflammatory properties. This study aimed to morpho-agronomically characterize 27 accessions of the collection of V. curassavica of the Active Germplasm Bank of Medicinal and Aromatic Plants of the Federal University of Sergipe. The experiment consisted of a randomized block design with three replications. The morphological characterization occurred in the field, by evaluating the variables plant height; canopy width; canopy shape; stem diameter; stem color; leaf blades width, length, and length/width ratio; leaves, petals, and sepals color. The agronomic characterization was performed using the essential oils extracted from dried leaves by hydrodistillation in a Clevenger apparatus. Leaves dry matter yield per plant and essential oil yield and content were also evaluated. The accessions showed great variation for leaves color, with different shades of green, which allowed separating them into dark green leaf and light green leaf plants. Plant height values ranged from 101.33 cm (VCUR-801) to 345.33 cm (VCUR-701). The clustering analysis of the traits revealed seven distinct groups. The accessions VCUR-103 and VCUR-001 were the most divergent, whereas the accessions VCUR-401 and VCUR-404 were the most similar. The morpho-agronomic characterization of V. curassavica accessions provides a correct description of the species by pointing out perspectives for genetic improvement, besides optimizing the conservation process.


INTRODUCTION
Brazil is rich in plant biodiversity (SOUZA et al., 2010). However, in recent years, its biodiversity has undergone accelerated destruction, mainly affecting the natural vegetation. This fact is due to agricultural expansion, fires, logging, extractivism, and uncontrolled urban growth. All these changes have posed a great threat to several plant species in the country (OLIVEIRA, 2010).
An alternative for the maintenance and conservation of endangered species is the formation of collections in Active Germplasm Banks, which holds base collections for the conservation of a broad plant genetic variability, enabling biodiversity maintenance (SILVA et al., 2012).
Varronia curassavica Jacq. (ex Cordia verbenacea DC.)] is a medicinal and aromatic plant native to Brazil, belonging to the Cordiaceae family (GASPARINO;BARROS, 2009). It occurs from Central America to southern Brazil (LORENZI; MATOS, 2008) in a wide range of habitats, such as beaches, restinga, forests, and cerrado (WANDERLEY et al., 2002). V. curassavica has stood out both in folk medicine and in the pharmaceutical industry, mainly due to the antiinflammatory substances present in its essential oil (PASSOS et al., 2007;PARISSOTO et al., 2012;PIMENTEL et al., 2012).
The effectiveness of V. curassavica is recognized by the National Sanitary Surveillance Agency (ANVISA). The plant is included in the List of the Unified Health System (SUS) and the National List of Medicinal Plants of Interest to the Unified Health System (RENISUS) (BRASIL, 2011). Due to its therapeutic properties, several studies have been developed aimed at the selection of superior accession for its agronomic aspects and chemical composition (VAZ et al., 2006).
In 2012, a V. curassavica collection was implanted in the Active Germplasm Bank of Medicinal and Aromatic Plants of the Federal University of Sergipe, containing accessions from the states of Sergipe and São Paulo, aiming to conserve the genetic variability of the species. An Active Germplasm Bank provides information on molecular, morphological, and agronomic variability. Moreover, it allows the knowledge about the diversity and instability of the essential oil chemical composition of the conserved accessions and their biological activities, consequently enhancing the exploitation of the species' genetic resources.
Besides the germplasm maintenance, the Active Germplasm Bank also provides the characterization of conserved accessions. Thus, all information regarding the morphological and agronomic traits, besides providing an "identity" for each accession, may be useful in the genotype selection process to compose breeding programs for this species (GOEDERT, 2007). Characterization is an essential activity in germplasm collections management and consists of describing, identifying, and differentiating accessions of the same species (BURLE; OLIVEIRA, 2010).
Morphological and agronomic characterization is the starting point for the knowledge of a plant species, especially when the objective is the selection of accessions of interest both for cultivation and for use in breeding programs (BLANK et al., 2017;OLIVEIRA et al., 2018). Therefore, this study aimed to morphoagronomically characterize 27 V. curassavica accessions of the Active Germplasm Bank of Medicinal and Aromatic Plants of the Federal University of Sergipe.

MATERIAL AND METHODS
The Varronia curassavica Jacq. (ex Cordia verbenacea DC.) collection from the Active Germplasm Bank of Medicinal and Aromatic Plants of the Federal University of Sergipe is implemented in the Research Farm "Campus Rural da UFS," located in the municipality of São Cristóvão, Sergipe, Brazil (lat. 11°00'S, long. 37°12'W). The collection contains accessions, obtained by vegetative propagation, from different locations in the states of Sergipe and São Paulo (Table 1).
The experiment consisted of a randomized blocks design with three replications. Each plot consisted of one plant, spaced at 2.0 m between plants and 3.0 m between rows. Fertilization was carried out every three months, using 5 L of bovine manure per plant. Monthly weeding was also performed.
The morphological and agronomic characterization of 27 V. curassavica accessions was carried out in June 2015.
The morphological characterization of the plants kept in the V. curassavica collection of the Active Germplasm Bank of Medicinal and Aromatic Plants of the Federal University of Sergipe was performed based on Blank et al. (2004). The following variables were evaluated: plant height (cm); canopy width (cm); stem diameter at 10 cm from the ground (cm); leaf blade length and width (mean of four fully expanded leaves randomly sampled from each plant), and leaf blade length/width ratio. Canopy width was measured by the mean between the smallest and the largest diameter. Qualitative evaluations were also performed for canopy shape (rounded, goblet, or irregular); stem, leaf vein, petals, and sepals color. The leaf area index was measured in four leaves per plant, using the LICOR leaf area meter, model LI-3 100C. Leaf color was determined by scanning four leaves collected from each plant on an HP 1005 MPF scanner. Colors were analyzed in R (red), G (green), and B (blue) patterns, using the color picker tool of Paint software for Windows. Six color measurements were performed on each side of the leaves (abaxial and adaxial). The mean of the color measurements for each side of each leaf formed the means for each plant. These means were used to transform the RGB color system into hexadecimal code, using the online software Webcalc, which represents the color pattern of the leaves of each plant. Sepals and petals colors were determined shortly after blooming, and stem color was determined visually.
The agronomic characterization was performed by analyzing the leaves dry matter and the essential oil content and yield. Leaves were collected and then weighed on an electronic scale. Afterward, they were dried in a forced-air circulation oven at 40 ºC ± 1 ºC for five days. After drying, the material was weighed to determine the dry matter.
The essential oil was extracted by hydrodistillation in a modified Clevenger apparatus for 140 minutes, coupled to a 3000 mL round bottom flask. For each accession, samples of 50 g of dry leaves were used for two liters of distilled water in triplicate.
The essential oil content and yield were calculated for each accession, following the equations: Content (%, v/m) = (Volume of essential oil extracted from the sample/leaves dry matter) x 100 Essential oil yield (mL/plant) = (content% x total plant dry matter) The quantitative data of the morphoagronomic characterization were subject to analysis of variance (ANOVA), and the means were compared by the Scott-Knott test (p≤0.05) with the aid of the statistical package Sisvar, version 5.6 (FERREIRA, 2011). From the morphological and agronomic characterization data, two multivariate analyses were performed, cluster analysis and principal component analysis (PCA), using the Statistica software. Subsequently, a dissimilarity matrix was constructed based on the morphoagronomic characteristics of each accession, according to their Euclidean distances. The dissimilarity matrix was simplified with dendrograms, using the Ward's clustering method.
Due to the differences of the units of the morpho-agronomic traits, data were standardized according to (REGAZZI, 2000), using the formula: Where: Zij = is the standardized value of observation i in variable j Xij = is the original value of observation i in variable j Xj = is the mean of the variable j S(Xj) = is the standard deviation of variable j

RESULTS AND DISCUSSION
V. curassavica accessions presented phenotypic variability for the evaluated traits. Accessions presented great uniformity for sepals and petals colors. However, some variations were identified for stem color, canopy shape, and leaf color (two shades of green) ( Table 2). A great variation was observed between the accessions for leaf color due to their different shades of green, which allowed separating them into dark green leaf and light green leaf plants (Table 3).
Considering the adaxial side, the accessions VCUR-002, VCUR-003, VCUR-004, VCUR-401, and VCUR-404 stood out for their dark green leaves, whereas the accessions VCUR-701, VCUR-303, and VCUR-105 stood out for their light green leaves. The RGB color blending indicates the specific color of the leaves sampled from the V. curassavica collection. The variation of the percentage of green determines the greenish shade of the leaves. A higher percentage of red in the three-color blend expresses a lighter shade of green, whereas a higher percentage of blue indicates a darker shade of green. In general, the accessions had a lighter shade of green on the abaxial side of the leaves.
For leaf area (LA), the accessions VCUR-402 and VCUR-601 revealed the highest means, with 144.38 and 117.48 mm², respectively, differing significantly from the others. Concomitantly, they were clustered with accessions that had the highest means for leaf length and width.
The accession VCUR-202 had the widest canopy width (CW), with 291.67 cm. The mean of the other accessions was 125.55cm. Such result is possibly due to the irregular canopy shape of this material. The accession VCUR-202 also had higher means for plant height (PH) and stem diameter (SD); conversely, this accession showed lower values for the other variables analyzed. The accessions VCUR-701 (345.33 and 19.33cm), VCUR-202 (295.00 and 18.33 cm), VCUR-201 (236.67 and 16.00 cm), and VCUR-001 (250.00 and 14.00 cm) had, simultaneously, the highest means for plant height (PH) and stem diameter (SD), respectively. The other accessions showed a mean of 9.04 cm of PH and 2.88 cm of SD. The accession VCUR-105 stood out with the highest essential oil content among the 27 accessions evaluated, with 3.20%, proving to be a promising accession to be used in breeding programs aimed at increasing essential oil production. For the essential oil yield, the accessions VCUR-505, VCUR-504, VCUR-101, VCUR-303, and VCUR-002 stood out with means  (Table 5).
The knowledge of the morpho-agronomic traits evaluated in germplasm collections allows better use of the species under conservation. The information generated in the characterization of the material also collaborated with the optimization of germplasm banks by eliminating accession duplication and reducing maintenance costs (BLANK, 2013).
Variability is important both for germplasm conservation and for use in breeding programs, as it increases the possibilities of the selection of materials that have promising and desirable attributes for the industry and the consumer market (CARVALHO et al., 2014). Values followed by the same letter in the column did not statistically differ from each other by the Scott-Knott test (P <0.05%).
The principal component analysis with two principal components explained 53.52% of the total variation ( Figure 2). The first principal component represented 29.40% of the total variation and was positively related to the variables R (AB) (r = 0.86); G (AB) (r = 0.91); R (AD) (r = 0.83); and G (AD) (r = 0.72). The second principal component represented 24.11% of the total variation and was negatively related to LL (r = -0.85), LW (r = -0.97), and LA (r = -0.94).

CONCLUSION
The results confirm the existence of phenotypic variability among V. curassavica accessions of the Active Germplasm Bank of Medicinal and Aromatic Plants of the Federal University of Sergipe. The information obtained in the present study assists the establishment of strategies to improve the species conservation and allows the exchange, extension, and availability of materials for breeding programs. The preserved accessions, by means of genetic breeding programs, may generate superior cultivars adapted to specific environments and with favorable traits, such as high yield of essential oils rich in the desired compounds.