Seeds characterization of wild species Jatropha peiranoi endemic of arid areas of Monte Desert Biome, Argentina

Industrial Crops & Products 141 (2019) 111796

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Seeds characterization of wild species Jatropha peiranoi endemic of arid areas of Monte Desert Biome, Argentina

T

Paula Paterlinia,1, Gloria Susana Jaimeb,1, Constanza Adenb, Cristina Olivaroc, ⁎ ⁎ María Inés Gómezb, Karina Cruzb, Ursula Tonellob, , Cintia Mariana Romeroa,b, a

PROIMI-CONICET, Av. Belgrano y Pasaje Caseros, T4001 MVB, San Miguel de Tucumán, Argentina Facultad de Bioquímica, Química y Farmacia, UNT, Ayacucho 471, T4000ILI, San Miguel de Tucumán, Argentina c Espacio de Ciencia y Tecnología Química, Centro Universitario de Tacuarembó, UdelaR, Uruguay b

A R T I C LE I N FO

A B S T R A C T

Keywords: Jatropha peiranoi Oil Clonal propagation Lipolytic enzyme

Jatropha peiranoi grows in the arid northern area of Monte Desert Biome in Argentina. This plant is adapted to low temperatures during the winter season. This study deeply describes the seed structure and oil characteristics as well as the clonal in vitro propagation of the plant in order to evaluate its potential as a new energetic crop. Wild seeds were harvested from Santa Maria Valley during the reproductive season. The color of its tegument showed a great variability given by the time of harvest and degree of ripening of the fruits. The endosperm was rich in oleosomes and protein bodies. The seed oil yield obtained was 33%. The lipid profile was linoleic acid (75%), oleic acid (15.6%), stearic acid (4%) and palmitic acid (5.9%). The oil was stable to the high temperatures, with a boiling point at 380 °C, without decomposition even at 400 °C. Lipolytic enzymes were detected in the seed-cake, with values of 84.5 U/g and 46.7 U/g for lipase and esterase, respectively. A protocol for in vitro clonal propagation was achieved employing nodal explants. For the shoot induction and proliferation, a concentration of BAP up to 0.2 mg/L caused an increase in the number of axillary shoots per explant. The optimum IBA concentration for the rooting was 0.2 mg/L. The plantlets were successfully acclimatized in soil. Currently, the field performance of clones with desirable traits is being evaluated. This work contributes to Jatropha species knowledge since Jatropha peiranoi is one of the adapted species to extreme environmental conditions that differs from other members of the genus.

1. Introduction Worldwide demands lead to the depletion of conventional and nonrenewable fossil fuels and to global warming through the emission of greenhouse gases. In this context, the sustainable production of nonfood oleaginous crops has become the alternative choice for ecofriendly biofuels production without affecting food security. Among the more important plant evaluated as non-renewable fuels, Jatropha curcas L. (Euphorbiaceae) was extensively studied. This is a perennial tree, native to México and Central America and distributed in tropical and sub-tropical areas. Initially, J. curcas, was considered the most promising biofuel feedstock. It was expected that it would have adaptability to a wide range of agro-climatic conditions and an oil quality reaching the international biodiesel standards (Atchen et al., 2008). However, in the last decade, this approach has changed in view of

low seed yield and poor oil quality obtained experimentally. In fact, in several countries recent attempts at commercial cultivation have failed because of low productivity in large scale cultivation programs (Mazumdar et al., 2018). The causes of failure of J. curcas as a biodiesel crop to meet the expected performance were low genetic variability, inadequate agricultural practices, seed toxicity and susceptibility to biotic and abiotic stress. For breeding high-yielding varieties as a sustainable alternative for producing competitive biofuel the Jatropha genetic resources were assessed. The analysis of a worldwide Jatropha accessions showed that Mesoamerican populations have significantly higher genetic diversity than African and Asian populations (Li et al., 2017), and the interaction effects of genotype and environment on seed yield were reported in different areas indicating a possible epigenetic effect (Kumar and Das, 2018; Valdes-Rodríguez et al., 2018; Rao et al., 2015; Sunil et al., 2011). The relatively small genetic diversity challenged the Jatropha

⁎

Corresponding authors at: Facultad de Bioquímica, Química y Farmacia, UNT, Ayacucho 471, T4000ILI, San Miguel de Tucumán, Argentina. E-mail addresses: [email protected] (U. Tonello), [email protected] (C.M. Romero). 1 Equal contribution in this work. https://doi.org/10.1016/j.indcrop.2019.111796 Received 17 April 2019; Received in revised form 5 September 2019; Accepted 16 September 2019 Available online 21 September 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

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(2013). The field-collected material corresponding to Jatropha peiranoi specimen was deposited at the LIL Herbarium (Miguel Lillo Herbarium), Tucumán (Argentina), as LIL 616.356.

breeding programs however the characterization of populations adapted to specific environments will enhance the perspectives of succeeding (Li et al., 2017; Martin and Montes, 2015; Lama et al., 2018; Trebbi et al., 2019). In Argentina, eleven species have been identified, including Jatropha curcas L. The J. curcas populations naturally growing in the warm subtropical Humid Chaco (Chaco Region) (Lourteig and O’Donnell, 1943), but not in arid areas where plants are affected by drought and frost damage in the winter season (Andrade et al., 2008; Ploschuk et al., 2014). However, in the Dry Chaco (Chaco Region), an environment with freezing winters, other species of Jatropha such as J. macrocarpa has been identified (Lourteig and O’Donnell, 1943). J. peiranoi L., is a subshrub (30–50 cm tall), with a woody succulent, more or less subterranean caudex, a deep root system, and decumbent branches that appear annually during the growing season. Fruits are trilocular capsules, explosively dehiscent when dry, with a single seed per locule. Is endemic to the northern area of Monte Desert Biome in western Argentina, with a very narrow distribution restricted to preAndean arid valleys (1700–1900 masl), along a longitudinal stripe of ca. 150 km length and 50 km wide, comprising the provinces of Catamarca, Tucumán and Salta. It grows in sandy soils with low content in organic matter and nutrients, in average temperature ranges of 37-13 °C in summer and 18–33 °C in winter, and annual mean rainfall of 200 mm restricted to the summer wet season (Lourteig and O’Donnell, 1943; Abraham et al., 2009). The seed oil content of 47% in a wild population of J. peiranoi located in Santa Maria Valley, Catamarca (27° 00’ S, 66° 14’W, 2200 m a.s.l.) was reported (Fracchia et al., 2016). The study of collected germplasm along diverse agro-climatic regions allowed the identification of desirable traits to be used in the crop improvement programs. In vitro micropropagation is a biotechnological tool that meets the demand for availability of uniform clones in short periods of time. The protocols developed for clonal propagation of elite genotypes will be used for large scale plantations. Tissue culture protocols using various explants were described for different species of Jatropha, including J. panduraefolia, J. intergerrima and J. curcas (Sujatha and Prabakaran, 2003; Rajore and Batra, 2005). The multiplication rate along the propagation phase obtained in these experiences was low and must be optimized. The clonal propagation of nodal explants was developed for J. curcas in order to maximize the number of plantlets and reduce the genetic variation (Sujatha et al., 2005; Datta et al., 2007). The characterization of Jatropha species adapted to environmental conditions that widely differ those from J. curcas will shed light on mechanisms involved in oil storage, fatty acid synthesis in seeds and to the adaptive response to abiotic stresses. In this work, a deep study of each part of the native plant, Jatropha peiranoi, was made considering especially the seed structure. The seeds were selected by phenotypic characteristics and an exhaustive evaluation of its parts was made.The oil distribution into the seeds was determined by different techniques and its chemical composition was analyzed. On the other hand, the clonal in vitro propagation of J. peiranoi under different culture conditions was evaluated also in order to analyze its potential for industrial application.

2.2. Morphological and histological seed characterization For the characterization, groups of 200 seeds were separated and classified considering the length, width, and thickness using a Vernier under a magnifying glass (10×). The seeds were classified into 5 groups (G1-G5) based on the different brown tones of the tegument. For the histological analysis, freehand cuts of endosperm and embryo were made. Sterilized seeds without caruncle were used, without cover or with seminal cover. They were fixed in FAA (Formaldehydeethyl alcohol-acetic acid-water 5:3:1:1), dehydrated and included according to the paraffin inclusion technique (Supplemental Material S1) (Dizeo de Strittmater, 1979; D’Ambrogio de Argüeso, 1986). Longitudinal and transversal cuts of 25 μm thickness were obtained, with Minot, Bausch & Lomb rotation microtome. Samples previously dewaxed, were stained applying differential stains, with Safranine 1% – Sudan IV 80% – Methylene blue 1%Safranina-Fast green saturated in 100% alcohol and Methylene BlueSudan IV for fats and oils detection (Conn, 1953). For starch detection, a Lugol solution was used following the technique of Ruzin (1999) and D’Ambrogio de Argüeso (1986). The permanent preparations were mounted in Balsam of Canada for later observation under an optical microscope. Microscopic photographs of preparations were taken with a TSVIEW 5.0 MP digital camera connected to an Olympus CKX41 Inverted Microscope. Transversal and longitudinal sections of seeds were prepared for examining the cellular structures. Oleosomes and protein bodies present in the seeds were observed by Transmission Electron Microscopy (TEM) Zeiss EM109 operated to 50 kV and Scanning Electron Microscopy (SEM) Zeiss Supra 55VP. All the observations were made in the Integral Center of Electron Microscopy (CIME), CONICET-UNT. 2.3. Jatropha seeds oil characterization For oil extraction, 8 mL of n-hexane was added to 2 g of the crushed seed, the sample was vortexed for 10 min and then centrifuged to recover the supernatant with the extracted oil. The biomass was continued treating with n-hexane until turbidity did not observe, in order to recover the residual oil. Next, it was centrifuged at 8000 rpm for 5 min. Then, the hexane was evaporated and the oil content was calculated according to the following: % Oil = (Oil weight/Initial seed weight) × 100. 2.3.1. Lipids profile determination The evaluation of the lipid profile was made by Gas Chromatography with flame ionization detector (GC-FID). First, 1 mg of oil sample was dissolved in 1 mL methylated mixture (HCl in MeOH) and heated in a dry bath at 100 °C for 2 h with sporadic agitation. After cooling, 1 mL of water was added and extracted with n-hexane (3 × 1 mL). The hexane extract was dried with anhydrous sodium sulfate and filtered. It was dried with N2 current and suspended in 1 mL of n-hexane (methyl esters of fatty acids-FAMEs). The FAMEs obtained were performed on a Shimadzu GC-2014 equipped with split/splitness injector and flame ionization detector (FID). A capillary column SE 52 (30 m × 0.25 mm × 0.25 μm) was used and the temperature program was: 150 °C for 2 min, (150–260) °C at 5 °C/min., 260 °C during 20 min. The temperature of the injector and the detector was 300 °C. The injection volume was 1 μL and was operated in split mode (100: 1), the carrier gas was He, 31 mL/min was the total flow and 0.55 mL/min was the flow of the column. The FAMEs were identified by direct comparison with pure standards and the individual percentages of them were made in relation to the total area of the chromatogram.

2. Material and methods 2.1. Plant material and botanical classification Fertile plants with flowers and fruits from Jatropha sp. were collected in Santa María Valley, Catamarca (Argentina), at 1885 masl, coordinates (66° 2 33’’ W–26° 41 16’’ S), during spring/summer season (October 2016–February 2017). For the identification of this species, the whole plant of Jatropha sp., with vegetative and reproductive parts, was herborized. The vegetal material was determined according to the taxonomic keys described by Lourteig and O’Donnell (1943) and Fernández Casas 2

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To calculate the composition of the fatty acids (%), the peaks were taken and the areas corrected. For this, the area was multiplied by a given factor for each particular fatty acid. The total area per sample indicates the total amount of fatty acids produced.

24 ± 1 °C and 16/8 h light/dark photoperiod until they reached an average 15 cm height. For the present study, the nodal explants were collected from these donor plants and then grown under field conditions.

2.3.2. Thermal analyses The thermogravimetric analysis (TGA) was carried out using a Shimadzu TGA-50 analyzer, under Nitrogen atmosphere at 20 mL/min. The analysis was carried out at different heating speeds (5, 10, 15 and 20°/min) from room temperature to 800 °C (in the platinum crucible). A sample of 8.267 mg was used, corresponding to a volume of 10 μL. The kinetics reaction in the differential thermal analysis was evaluated (Kissinger, 1957; Agrawal and Chakraborty, 2013).

2.5.2. Basal medium and culture conditions In all experiments, Murashige and Skoog 1X (MS) basal medium was prepared according to manufacturer’s instructions (M519 PhytoTechnology Laboratories™), supplemented with sucrose 3% (w/v) and 0.65% agar. The plant growth regulators were added to the medium as specified below. The pH was adjusted to 5.7 ± 0.1 using KOH 1N. The media were dispensed in bottles and autoclaved at 15 psi at 121 °C for 20 min. The sterilized media (20 mL) was distributed in tubes (15 × 2.5 cm). All the cultures were kept in a growth room at 24 ± 2 °C with 16 h photoperiod with a flux density of 30 μmol m−2 s−1 by fluorescent tubes of daylight.

2.4. Determination of lipolytic enzymatic activity in the seed-cake To evaluate the enzymatic activity, the seed-cake obtained after the extraction of the oil was used. The n-hexane present in the seed-cake was evaporated and then it was placed in an oven at 30 °C until constant weight. Then, the dry biomass was used for enzymatic determination. Esterase and lipase activities were measured using p-nitrophenyl acetate, C2 (p-NPA) and p-nitrophenyllaurate, C12 (p-NPL) 1 mM as substrates respectively. For enzymatic determination, 0.05 g of dry seed-cake of Jatropha peiranoi seeds was added to 1 mL of buffer A containing: buffer phosphate 100 mM (pH 7), gum arabic 0.1% (w/v) and Triton X-100 0.4% (w/v) (Winkler and Stuckmann, 1979). The reaction mixture was shaken at 150 rpm and 37 °C. After the incubation, the mixture was centrifuged at 10,000 rpm for biomass separation. The p-nitrophenol (p-NP) released as a result of enzymatic hydrolysis was estimated spectrophotometrically at 405 nm. One unit of enzyme activity (U) was defined as the amount of biocatalyst that released 1 μmol of p-NP per minute under the standard assay conditions.

2.5.3. Explant surface sterilization and culture establishment For surface sterilization, the nodal explants (1.0–1.5 cm in length) were first washed thoroughly under running tap water for 20 min. Then they were transferred to ethanol 70% for 1 min, rinsed three times with sterile distilled water and soaked in a sodium hypochlorite solution 10% for 10 min. Following repeated washes with sterile distilled water, each sterilized explant was trimmed in pieces of 1 × 1 cm and transferred to culture tubes (15 × 2.5 cm) containing 20 mL of culture medium.

2.5. In vitro clonal propagation of nodal explants of Jatropha peiranoi

2.5.4. Shoot induction and proliferation For multiple shoot induction, nodal explants were cultured on MS medium supplemented with 0–1.0 mg/L of BAP (6-benzylamino purine) (Table 2). Data were collected after four weeks of culture initiation. The regenerated shoots were cut into segments each with a single node and transferred to fresh media every 4–6 weeks and the multiplication rate per explant was calculated.

2.5.1. Seed germination and explant preparation To perform the in vitro culture procedures, the seeds from mature fruits were classified into five groups according to the color and appearance of the tegument (Table 1). The sterilization of the seeds was made according to Comfort et al. (2018). The seeds were washed with water to remove the dirt and then sterilized by immersion in 70% absolute ethanol (v/v) and 10% (v/v) sodium hypochlorite (NaOCl) during 10 min, followed by sterile water. Then, the seeds were removed from the seed coats and the endosperm obtaining the embryo for evaluation. A study of the morphology of the seeds and the size of embryos was made. The selected seeds from Jatropha peiranoi were sown in pots using sterile sand soil. The seedlings were grown in a greenhouse at

2.5.5. In vitro rooting and acclimatization Well-developed shoots (1.5–2.0 cm) with 3–4 leaves were transferred to a root induction media consisting of full strength MS medium with IBA (indole-3-butyric acid) at different concentrations (Table 3). Percentage of root induction, number of roots per shoot and length of roots after four weeks of culture were recorded. Plantlets with developed shoots and roots were removed from the culture medium and washed with running tap water. Then, they were transferred to plastic pots containing sterile sand soil and covered with transparent plastic bags and maintained in a greenhouse at 24 ± 2 °C under daylight fluorescent tubes (16/8 h photoperiod). After two weeks, the plastics bags were opened in order to acclimatize the plantlets to field conditions.

Table 1 Biometric data of the seeds of J. peiranoi and measurements of the embryo length corresponding to each group. The obtained data represent the averages of measurements and the standard deviations made in 200 seeds of each group and their respective embryos. n = 200 Analysis of the variance p ≤ 0.05 was performed. Averages that do not share the same letter are significantly different. NMLB: Not marbled light brown. MLB: Marbled light brown. DB: dark brown. NMGB: not marbled greyish brown. MGB: marbled grayish brown. Groups Measurements (cm)

1 NMLB

2 MLB

3 DB

4 NMGB

5 MGB

Seeds

Length Width Thickness

0.85 ± 0.06a 0.44 ± 0.02a 0.33 ± 0.02a

0.93 ± 0.06a 0.47 ± 0.03a 0.37 ± 0.02a

0.92 ± 0.04a 0.46 ± 0.03a 0.35 ± 0.02 a

0.92 ± 0.06a 0.46 ± 0.03a 0.35 ± 0.02a

0.93 ± 0.06a 0.47 ± 0.06a 0.36 ± 0.0 a

Embryos

Length

0.45 ± 0.12a

0.64 ± 0.16a

0.71 ± 0.08b

0.74 ± 0.10b

0.72 ± 0.07b

3

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2.5.6. Statistical analysis Statistical analysis was performed using Minitab (Minitab, Inc.) software version 14 for Windows. Experiments were set up in randomized block design and each experiment had three replicates; each of which consisted of 10 culture tubes. The analysis of variance (ANOVA) was carried out to detect the significance of differences among the treatment means (plant growth regulators concentrations). The treatment means were compared using Tukey test at the 5% probability.

Table 2 Effect of BAP (6-benzylamino purine), on shoot induction from nodal explants of J. peiranoi after four weeks of culture. Each mean is based on three replicates, each consisted of 10 culture tubes. The letters indicate significant difference between means (P < 0.05); comparison by Tukey test. BAP (mg/L)

Percentage of response d

0.1 0.2 0.5 1.0

Number shoots/explant 4.2b 3.9b 2.3a 1.9a

98 85c 40b 32a

3. Results and discussion 3.1. Botanical classification

Table 3 Effect of IBA (indole-3-butyric acid), on rooting of shoots of J. peiranoi after four weeks of culture.Each mean is based on three replicates, each consisted of 10 culture tubes. The letters indicate significant difference between means (P < 0.05); comparison by Tukey test. IBA (mg/L)

Percentage of root induction

Number roots/ shoots

Root length (cm)

0.1 0.2 0.5 1.0

40b 85d 72c 19a

1.2a 5.6c 2.4b 0.9a

5.4c 9.5d 4.7b 1.8a

The collected species corresponds to Jatropha peinaroi Lourteig & O’Donell (Fig. 1A) scientific name currently accepted for the Southern Cone Flora according to Zuloaga et al. (2008). It belongs to the Euphorbiaceae family; it is an endemic plant of the provinces of Catamarca, Salta, Tucumán and grows between 1700 and 1900 masl (Lourteig and O’Donnell, 1943). Jatropha peiranoi is a shrub plant, herbaceous in appearance and creeping. It grows forming tufts, in patches up to 50 cm in height. It has a fleshy stem, 4–7 mm in diameter, light green to purple and branched from the base. Also present, an underground stem, thick rhizome from 30 to 50 mm of diameter.

Fig. 1. Jatropha peiranoi Lourteig & O’Donell. A-Vegetative aspect of the plant in its natural habitat. B1 and B2- Detail of petioles with foliar expansions and glandular structures. B3-Details of mucronate leaf blade. C-Cimosa inflorescences with fructified male and female flowers. Variation of flower colors, light pink to fuchsia. D1Capsule fruits (1×). D2-Seeds (10×), variability of colors is observed. D3-Close inmature fruit (20×). D4-Ripe fruits close to opening or dehiscence (20×). 4

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Fig. 2. Jatropha peiranoi Lourteig & O’Donell. A-Morphological aspect of the seeds seen at the magnifying glass. Caruncle or eleosome with whitish edges are observed the raphe (rf) and hilum (h). B-SEM microphotography. Details of the branches of the caruncle or eleosoma (Ca).

are dispersed through the sudden dehiscence of the fruit (ArandaRickert, 2011). These described characters allowed the identification of the harvested plants as J. Peiranoi Lourteig & O'Donell according to Lourteig and O’Donnell (1943), and Fernández Casas (2013).

The petioles are of 1–7 mm, glabrous, channelized, with leaf expansions of varied form, usually lanceolate, with notable stipitate glands (Fig. 1-B1). The plant has leaf coriaceous foliar 3–5 batches, with a rounded base with hirsute hairs (Fig. 1-B2). The leafs edge are yellowish-green softly crenate (Fig. 1-B3). Jatropha peiranoi has unisexual flowers (Fig. 1C). Male Flower: Sepals 5, ovate-lanceolate, acuminate, hairy, glandular-ciliated border. Opaque petals yellowish, light pink and dark internally, pubescent. Disk: 5 free glands. Stamens 10, monadelphous arranged in 2 cycles. Female Flower: Sepals 5 obovate-lanceolate, acuminate, glandularciliate. Petals whitish, yellowish, pink to fuchsia internally and glandulose-stipulated. Disk: 5 free glands. Ovary glabrous or pubescent, and Styles 3. Fruits capsule, trigamocarpelar, trilocular, one seed per locule, green at the immaturity and brown at maturity (Fig. 1-D1 and 1D2).The fruit is an ovoid capsule, pubescent, schizocarp type, formed by three carpels, each containing a seed, green to the immature state and brown at maturity (Fig. 1-D3 and 2-D4). The dehiscence of this plant is explosive or ballistic, it is a type of autochory in which the seeds

3.2. Morphological and histological characterization of J. peiranoi seeds The seeds dimensions were referred to average values of 200 seeds of each group. The longitudinal size varied between 8.5 and 9.3 mm, the thickness between 3.3 and 3.7 mm and the width between 4.4 y 4.7 mm. The embryo lengths presented values between 0.45 and 0.74 mm (Table 1). The seeds are oval or ellipsoid with hard consistency, compressed ventral side and dorsally convex plane. The seminal surface was slightly smooth with a soft satiny gloss and variable colours (Fig. 2A). Close to the micropyle, was observed a superficial excrescence, the caruncle or eleosome, oil-rich divided into nine branches with whitish edges, oriented towards the dorsal side (Fig. 2A and B). The seed morphology consisted of a seminal coat or testa, the 5

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Fig. 3. A-Jatropha peiranoi Lourteig & O’Donell. Stereoscopic microscopy. A1Longitudinal lateral section of the seed. Testa or tegument (t), endosperm (e) and cotyledons (c) are observed. A2-Middle longitudinal section: testa (t), endosperm (e), cotyledons (co) and radicle (r). A3-Without test, (e), (c) and (r). B- Scanning electron microscopy (SEM). B1-Transversal cut, (t), (e) and (co). B2Medium longitudinal section. (t), (e) and (co). B3-(t), (e), (c) and (r). B4- Detail of the radicle (r). C-Microphotographs obtained by optical microscopy. C1 and C2-Seed transversal cut showing internal structure: external tegument (a), internal tegument formed by exothegmen or mesophile (b) and endotegmen (c), remains of nucellus (d) and endosperm with oleosomes and protein bodies (e).

seed coat anatomy of Jatropha genus. In this work, the histological and ultrastructural description of transversal and longitudinal sections of J. peiranoi seed is described for the first time. The tissue sections observed in the tegument were similar to the one described by Esau (1982) for Ricinus communis (Flores and de Jesús, 2011), a specie of the Euphorbiaceae family. The tegument colour showed different brown tones (brown marmoreal dark brown, chestnut seeds, greyish chestnuts, lightly marbled and light brown to greyish brown seeds with little marbled to non-marbled) and according to that, the seeds were classified in five groups (G1-G5) (Table 1). The development of seeds is a complex process that involves reserve compounds accumulation and tolerance to desiccation. These processes require the concerted action of several signalling pathways involved in genetic programs associated

endosperm and the embryo, which is mainly composed of the radicle (embryonic axis) and cotyledons. In longitudinal sections, the seminal coat, endosperm and two cotyledons were observed inserted in a short and straight embryonic axis. In the cross section, the same structural and seminal compositions were observed (Fig. 3A and B). The outer tegument or testa, formed by a slightly rough cuticle and pigmented columnar macroesclereids arranged radially, confers to the seed a dark brown color. The internal tegument showed an exotegmen or mesophile, formed by collapsed parenchymal cells, and an endotegmen, with enlarged sclerosed radially and tangentially arranged columnar esclereids cells. The internal tegument is upholstered by spongy-looking nucellus remains, surrounded endosperm (Fig. 3C). In these seeds a large volume is occupied by the endosperm. There are few reports on 6

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Fig. 4. Cross section of endosperm of the seed. A1-Sudan IV-Safranin-Methylene Blue stain. A2 and A3-Sudan IV-Safranin. A4-Sudan IV-Methylene blue. Oleosomes, protein bodies (PB) and drusen (Dr) (Ca oxalate crystals) are observed. B1 and B2-Microphotographs obtained by scanning electron microscopy (SEM). Endosperm of the seed in different degrees of magnification, observing lipid bodies (LB) and protein bodies (PB). C-Microphotographs obtained by transmission electron microscopy (TEM) from ultrathin sections of endosperm, observing lipid bodies (LB), medium lamellae (Ml and cell wall (Cw). Table 4 Fatty acids composition of the triglyceride in different species of Jatropha compared with Jatropha peiranoi. A comparison is also made with soybean and olive oil. Compound

Palmitic acid Stearic acids Oleic acid Linoleic acid

Composition (%)

C C C C

16: 18: 18: 18:

0 0 1 2

Jatropha peiranoi

Jatropha curcas

Jatropha macrocarpa

Jatropha hieronymi

Jatropha excise

Soy

Olive

5.9 4 15.6 74.5

14–15 3.7–9.8 34.3–45.8 29–44.2

11–12 3–4 27–39 42–55

11–14 7–9 20–30 45–58

8.2 5.68 18.36 66.49

7–11 3–6 22–34 50–60

11.5 2 78 7

the same cellular characteristics as endospermic cells in terms of its content rich in oleosomes (Fig. 4B and C).

with different metabolic pathways. Expression genetic libraries were constructed for Jatropha curcas, corresponding to different stages of embryo development (Chen et al., 2011; Silva Pinto et al., 2018). Each of these stages is characterized by physiological events, and are associated with changes in the size, and dry weight of the seed. The analysis of genes expression profiles showed that in the final stage of development of the embryo the synthesis of proteins and oils is completed, related to an increase of the dry weight and decrease of the water content in the seeds (Jiang et al., 2012; Zavala-Hernández et al., 2015). The differences found in the embryos of J. peiranoi allow us to hypothesize that the changes observed in the appearance and colour of the tegument could be associated with different stages of seed development, associated with changes in the length of the embryo (Table 1). These stages ensure first the accumulation of reserve substances in the storage structures of the seed before the embryo develops to reach physiological maturity. For this reason, the seeds of G4 were selected for germination. Histochemical studies of the seeds were made also. These revealed the presence of fats and oils in the endosperm and embryo of J. peiranoi seeds, which were stained orange-yellow, with Sudan IV alcoholic saturated solution. On the other hand, the presence of starch wasn´t detected with Lugol's aqueous solution (Iodine and Potassium Iodide, IKI). (Ruzin, 1999; D’Ambrogio de Argüeso, 1986) (Fig. 3C). The cells of endosperm tissues contain about 13–15 oleosomes or oil bodies, grouped spheroidal structures measuring between 2.5 and 4.5 μm in diameter. Adriano-Anaya et al. (2014) describe similar sizes for J. curcas. They reported oleosomes from 2.18 μm to 4.15 μm in diameter, in a range of 13 to 15 per cell and with spatial arrangement grouped and dispersed in 85 genotypes of J. curcas populations studied. In Jatropha peiranoi seeds, the endospermic cells (reserve parenchyma) are observed in different degrees of magnification with oleosomic and protein structures inside, occupying all the cell volume. The embryo has

3.3. Jatropha seeds oil characterization After the extraction using n-hexane a performance of 33% of oil was obtained. To analyze the lipid profile of Jatropha peiranoi oil the fatty acid composition was analyzed by GC-FID. The analysis showed that the triglyceride of Jatropha is composed of linoleic acid (75%), oleic acid (15.6%), stearic acid (4%) and palmitic acid (5.9%) (Table 4). Table 4 showed also a comparison between fatty acid composition of J. peiranoi with others vegetable oils of the same genus but different species such as J.curcas (Berchmans and Hirata, 2008), J.excisa (Clérici et al., 2013), J. macrocapa and J. hieronymi (Falasca and Ulberich, 2008) and other vegetable oils such as soybean (Mayo, 2013) and olive (Kostik et al., 2013). A variation in the composition of fatty acids can be observed. Particularly, the seeds of Jatropha species have a very wide range regarding the oil content, about from 18 to more than 40% as a dry matter of whole seed. This value varies depending on the ecotype, pedoclimatic condition, seed storage and harvest times (Verma and Verma, 2015; Wassner et al., 2016; Senou et al., 2017). In our case, more studies are being made in J. peiranoi to determine the influence of these variables in the oil composition. To characterize the process of thermal degradation of the oil, the thermogravimetric analysis was carried out, which allowed measuring the mass loss of the oil sample used (8.267 mg), as a function of the temperature under a controlled inert atmosphere of nitrogen. Fig. 5 presents the TGA of Jatropha peiranoi oil at different temperatures. Fig. 5A shown three steps of mass loss at a velocity of 5 °C/min while at higher speeds (15 °C/min and 20 °C/min), only one step is observed (Fig. 5B–D). The process of decomposition ending in the range between 7

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Fig. 5. Thermogravimetric analysis of Jatropha peiranoi oil. The TGA was carried out at different heating speeds (5, 10, 15 and 20 °C/ min) from room temperature to 800 °C.

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Lipolytic enzymes from different oilseeds have been studied, such as flax seeds, sunflower seeds, Jatropha curcas, among others (Staubmann et al., 1999; Sadeghipour and Bhatla, 2003; Barros et al., 2010). However, lipase activity in Jatropha peiranoi seeds has not been studied until now. The seed-cake of J. peiranoi showed both enzymatic activities, with an esterase activity of 4.23 U/L and lipase of 2.35 U/L and specific activity of 84.5 U/g and 46.7 U/g respectively (Fig.6). In this way, the obtaining of lipolytic enzymes from the seed-cake offers the possibility of proposing a production cycle of biofuel such as biodiesel by transesterification of J. peiranoi oil catalyzed by lipases from the plant itself. This type of biotechnological platform has been proposed in recent years to give added value to technologies with a commercial application (Zhu et al., 2016). In our case, this platform is generated from an oleaginous vegetable species native to our region, not edible, for application in several industrial areas.

Fig. 6. Lipolytic activities of wet biomass from seed cake obtained after the oil extraction. Esterase and lipase activities were determined using p-NPA (p-nitrophenyl acetate, C2) and p-NPP (p-nitrophenyllaurate, C12) respectively.

466.08 °C and 480.30 °C, depending on the heating velocity. This is due to the volatility of the oil components increases with the temperature. The total mass loss was approximately 100% that suggests that a process of boiling occurred. The TGA curve shows that the thermal stability of Jatropha peiranoi was around to 370 °C. The oil began to boiling under a nitrogen atmosphere without decomposing even until 400 °C when the heating speed was higher. The peaks of the first derivative of the TGA were used to determine the kinetic parameters using the Kissinger method, resulting in Ea = 118 kJ/mol and pre-exponential factor of Arrhenius A = 6.34 108 K mol. It was found that the decomposition process has a kinetic of order 0, with a correlation coefficient of R2 = 0.9957, which means that the kinetics of the reaction is independent of the oil concentration (see Supplemental material S3). The results of the thermal stability of J. peiranoi oil were similar to observed in J. curcas L. oil from several Brazil crops (Freire et al., 2009). However, some differences may be observed, which could be due not only the differences in the fatty acid composition of both species but also the age of the seeds or the storage time of its.

3.5. Micropropagation of J. peiranoi 3.5.1. Shoot induction and proliferation Explants cultured in MS medium without cytokinin failed to induce shoot proliferation. MS medium supplemented with different concentrations of BAP (0–1.0 mg/L) resulted in the induction of axillary shoots (Table 2). The frequency of responding explants and the number of axillary shoots per explant increased with an increased in the concentration of BAP between 0.1 and 0.2 mg/L. These results indicated that BAP played an important role in the induction of multiple shoots. However, BAP at higher concentrations not only reduced the number of shoots formed but also had an effect on their growth since the length was reduced. After four subcultures (120 days) in fresh media, the mean multiplication rate was 4.5 shoots per explant (Fig. 7A and D). The effect of BAP on the induction of shoot in nodal explants has been reported earlier for J. curcas alone or in combinations with other cytokinins (Kalimuthu et al., 2007; Thepsamran et al., 2008; Sujatha et al., 2005; Behera et al., 2014). The regenerated shoots of J. peiranoi showed a similar multiplication rate than those reported for J. curcas (Sujatha et al., 2005).

3.4. Lipolytic enzyme activity in the byproduct cake Jatropha seed oil is the most important product evaluated for biofuel or lubricant (Kumar and Sharma, 2008). However, different parts of Jatropha plant contain a range of interesting metabolites and bioactive compounds (Moniruzzaman et al., 2016). After oil extraction, the resulting seed-cake could be an excellent protein source (León-Villanueva et al., 2018). Seed-cake is a source of enzymes, which provides a potential added value of great commercial interest. The use of biocatalysts or enzymes allows the application of biocatalysis processes at the industrial level, which contributes to the development of green industries in the process. That is why being able to obtain enzymes as a byproduct of obtaining Jatropha oil makes it a more profitable product. It is the case of the recovery of lipolytic enzymes such as esterase or lipase. These enzymes are present in plant biomass mainly in oilseeds (Barros et al., 2010).

3.5.2. In vitro rooting and acclimatization For rooting, shoots were excised from proliferated cultures and transferred to full strength MS medium supplemented with different concentration of IBA (0.1, 0.2, 0.5, 1.0 mg/L). Analysis of variance revealed a significant effect on the frequency of cultures showing root regeneration, number of roots/shoot and mean root length (Table 3). The addition of 0.2 mg/L of IBA in the media produced 5.6 roots/shoot in 85% of cultures after four weeks. However, a higher concentration of IBA (0.5–1 mg/L) inhibited the response of the plantlets. The effect of IBA for rooting in Jatropha curcas has been reported previously, and a broad range of IBA concentration was used (Datta et al., 2007; Kaewpooa and Te-chatob, 2009; Rajore and Batra, 2005; Kumar et al., 2010). J. peiranoi regenerated shoots present a similar behavior than

Fig. 7. In vitro clonal propagation of nodal explants of Jatropha peiranoi. A, B-Induction of shoots from nodal explants. C, D-Response of shoots and micropropagation on MS medium with BAP. E-In vitro rooting of shoots. F-Ex vitro establishments of the plantlets. 9

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the observed in J. curcas and showed a well-developed root system after 4–6 weeks on culture tubes. Plantlets with fully expanded leaves and well-developed roots were hardened using sterile sand soil in a greenhouse for four weeks. About 78% of acclimatized plantlets survived in field conditions and no detectable variation was observed with respect to morphology and growth characteristics (Fig. 7E and F).

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4. Conclusions This work represents the first stage for the classification, characterization, and in vitro propagation of a wild species from arid areas of Monte Desert Biome, Argentina. The histological and ultrastructural characterization of seed anatomy was reported. The seed oil yield and oil composition were obtained, and the lipolytic enzymatic activity (lipase and esterase) in the seed-cake was identified adding value to this byproduct. The development of a method for the availability of J. peiranoi plantlets through axillary shoot proliferation was useful for large scale multiplication of this species of Jatropha genus, tolerant to abiotic stresses. The morphological, histological, physicochemical, nutritional and enzymatic knowledge of J. peiranoi, contribute to broadening the spectrum of non-edible raw materials useful for industrial application. Acknowledgements This work was supported by the following Argentine research grants 26D-616 and 26E-631PIUNT (UNT) and PIP 0677/2015CONICET; University Volunteer Program, Res. N° 4136/2017 SPU and San Pablo-T University grant. Special thanks to Professors Romero and Fuenzalida for the help with the seeds harvest. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2019.111796. References Abraham, E., del Valle, H.F., Roig, F., Torres, L., Ares, J.O., Coronato, F., Godagnone, R., 2009. Overview of the geographic of Monte Desert Biome (Argentina). J. Arid Environ. 73, 144–153. Adriano-Anaya, M.L., Gómez-Pérez, J.A., Ruiz-González, S., Vázquez-Ovando, J.A., Salvador-Figueroa, M., Ovando-Medina, I., 2014. Oleosomas de semillas de Jatropha curcas L. como estimadores de diversidad en poblaciones del Sur de México. Grasas y Aceites 65 (3). Agrawal, A., Chakraborty, S., 2013. A kinetic study of pyrolysis and combustion of microalgae Chlorella vulgaris using thermo-gravimetric analysis. Bioresour. Technol. 128, 72–80. Andrade, G.A., Caramori, P.H., Souza, F.S., Marur, C.J., Ribeiro, A.M.A., 2008. Temperatura mínima letal para plantas jóvenes de pinhão-manso. Bragantia 67 (3), 799–803. Aranda-Rickert, A., 2011. Ecología de la dispersión de semillas por hormigas en Jatropha excisa Griseb. (Euphorbiaceae) (Doctoral Dissertation). Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Atchen, W.M., Verchot, L., Franken, Y.J., Mathjis, E., Singh, V.P., Aerts, R., Muys, B., 2008. Jatropha biodiesel production and use. Biomass Bioenergy 32, 1063–1084. Barros, M., Fleuri, L.F., Macedo, G.A., 2010. Seed lipases: sources, applications and properties—a review. Braz. J. Chem. Eng. 27 (1), 15–29. Behera, L.K., Nayak, M.R., Nayak, D., Jadeja, D.B., 2014. In vitro mass multiplication of Jatropha (Jatropha curcas L.) through axillary bud culture. J. Appl. Nat. Sci. 6, 189–192. Berchmans, H.J., Hirata, S., 2008. Biodiesel production from crude Jatropha curcas L. seed oil with a high content of free fatty acids. Bioresour. Technol. 99 (6), 1716–1721. Chen, M.S., Wang, G.J., Wang, R.L., Wang, J., Song, S.Q., Fu Xu, Z., 2011. Analysis of expressed sequence tags from biodiesel plant Jatropha curcas embryos at different developmental stages. Plant Sci. 181, 696–700. Clérici, S., Aranda, J., Martínez, P., Torchán, C., Baravalle, F., 2013. Caracterización morfológica y citogenética de Jatropha excisa, en la ecorregión Chaco Seco de la provincia de Catamarca. Biología en Agronomía 3 (1), 24–36. Comfort, A.C., Cyprian, O.C., EziucheAgaba, O.C., 2018. In vitro plant regeneration from mature embryo explants of Jatropha curcas L. (A Biodiesel Plant) on two standard basal nutrient media. Am. J. Plant Physiol. 13 (1), 23–35. Conn, H.J., 1953. Biological Stain. A Handbook on the Nature and Uses of the Dyes

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Diversity between Jatropha curcas L. accessions based on oil traits and X-ray digital images analysis from it seeds. Crop Breed. Appl. Biotechnol. 18, 292–300. Staubmann, R., Ncube, I., Gübitz, G.M., Steiner, W., Read, J.S., 1999. Esterase and lipase activity in Jatropha curcas L. seeds. J. Biotechnol. 75 (2), 117–126. Sujatha, M., Makkar, H.P.S., Becker, K., 2005. Shoot bud proliferation from axillary nodes and leaf sections of non-toxic Jatropha curcas L. Plant Growth Regul. 47, 83–90. Sujatha, M., Prabakaran, A., 2003. New ornamental Jatropha hybrids through interspecific hybridization. Genet. Resour. Crop Evol. 50, 75–82. Sunil, N., Sujatha, M., Kumar, V., Vanaja, M., Basha, S.D., Varaprasad, K.S., 2011. Correlating the phenotypic and molecular diversity in Jatropha curcas L. Biomass Bioenergy 35, 1085–1096. Thepsamran, N., Thepsithar, C., Thongpukkdee, A., 2008. In vitro induction of shoots and roots from Jatropha curcas L. explants. J. Hortic. Sci. Biotechnol. 83, 106–112. Trebbi, D., Ravi, S., Broccanello, C., Chiodi, C., Stevanato, P., 2019. Genomic resources and marker-assisted selection in Jatropha curcas. Jatropha, Challenges for a New Energy Crop. Springer, Singapore, pp. 145–160. Valdes-Rodríguez, O.A., Pérez-Vazquez, A., Palacios-Wassenaar, O., Sanchez-Sanchez, O., 2018. Seed diversity in Native Mexican Jatropha curcas L. and their environmental conditions. Trop. Subtrop. Agroecosyst. 21, 521–537.

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