INDIRECT ORGANOGENESIS AND ESTIMATION OF NUCLEAR DNA CONTENT IN REGENERATED CLONES OF A NON-TOXIC VARIETY OF Jatropha curcas

Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A.C., Unidad Sureste Mérida, Yucatán, México Email: glopez@ciatej.mx Tel: 523333455200 Ext. 4028 Center for Functional Ecology, Department of Life Sciences, University of Coimbra, Coimbra, Portugal. Instituto de biotecnología y ecología Universidad Veracruzana, Campus para la Cultura, las Artes y el Deporte, Xalapa, Veracruz, México. División de Ingeniería y Ciencias Exactas, Universidad Anáhuac Mayab, Mérida, Yucatán, México Agroindustria Alternativa del Sureste LODEMO Group, Mérida, Yucatán, México. *Corresponding author


INTRODUCTION
The species Jatropha curcas L. belongs to the family Euphorbiaceae and is believed to have originated in Mexico and Central America (Contran et al., 2013).The importance of this crop resides mainly in the fact that its seeds contain approximately 40% of oil with a chemical composition appropriate for transformation into high quality biodiesel by transesterification (Li et al., 2016;Soares et al., 2016).This species also presents alternative applications, such as its use in the recovery of areas degraded by mineral exploitation and by deforestation of devastated areas, which gives added value to its industrial use (Carels, 2013;Sabandar et al., 2013;Dias et al., 2012).In addition, the latex has been attributed with medicinal properties, in particular in the treatment of skin lesions (Sabandar et al., 2013).The biofuel obtained from this species is ecological and biodegradable, which has attracted worldwide attention as an alternative source of sustainable energy for the future (Baran Jha et al., 2007).Given the importance of the species in Mexico, the varieties Gran Victoria, Doña Aurelia, Don Rafael (Zamarripa and Solís, 2013) and the ALJC01 (Góngora-Canul and Martínez-Sebastian, 2016) have been registered in the National Service of Seed Inspection and Certification (SNICS -for its Spanish acronym), the latter being of particular agronomic importance given that it is a low, monoecious shrub with an open canopy and profuse branching that presents an average of 90 bunches of fruit per plant, an oil content of approximately 40%, a yield of 2.5 tons at a planting density of 2000 plants per ha and presents a phorbol esters concentration of 0.044 ± 0.003 mg/g (Sacramento-Rivero et al., unpublished data), according to Makkar et al. (1997) is considered potentially toxic when the concentration of phorbol esters is > 0.11 mg/g.So far only a non-toxic variety has been reported from Papantla region of the state of Veracruz in Mexico, suitable for human consumption (Makkar et al., 1997).
In J. curcas, in addition to the establishment of genetic improvement programs through the use of agrobiotechnological methods aimed at increasing seed and oil yields, it is also important to satisfy the demand and conservation for plant material from elite varieties, in this respect, no much work has been done on germplasm conservation (Kumar and Sharma, 2008); this creates the need to develop techniques of massive multiplication and conservation of the species (Panghal et al., 2012) to get the demand on a large scale and ensure easy supply of this elite material (Kumar et al., 2011a;kumar et al., 2015).Within this context, micropropagation protocols in J. curcas have been reported in toxic varieties, however, regeneration efficiency was observed in toxic cultivars compared to non-toxic cultivars (Kumar 2011, Kumar et al., 2011a, 2011b), this agrees with what was reported by Sharma et al. (2011) who have reported that nontoxic varieties of J. curcas are less sensitive to in vitro organ formation.Another factor that affects in vitro regeneration is the genotype / cultivar (Feyissa et al., 2005;Landi and Mezzetti, 2006;) in J. curcas, several authors report the high dependence of the genotype on in vitro regeneration (da Camara Machado et al., 1997;Kumar 2008;Kumar and Reddy 2010;Kumar et al., 2010) this behavior may be related to mechanisms and the endogenous content of the metabolism of plant growth regulators (sharma et al., 2011) which include morphogenesis using different concentrations of growth hormones, followed by optimization of the conditions for rapid regeneration (Gangwar et al., 2016), the induction of adventitious shoots from the petiole (Liu et al., 2015), the use of different types of explants and plant growth regulators for direct and indirect morphogenesis (Verma and Verma, 2015;Ali et al., 2015), and the induction of adventitious shoots and the development of plantlets from petiole explants (Liu et al., 2016), There are reports of non-toxic varieties in Jatropha curcas in which, regeneration was obtained by indirect organogenesis from leaf (Sujatha et al., 2005) and petiole (Kumar et al., 2010(Kumar et al., , 2011a)).
Plant tissue culture is a group of techniques applied in vitro, which includes the culture of plant tissue in an artificial medium under controlled environmental conditions (Levitus et al., 2010) with the aim to generate micropropagation protocols that can genetically improve of the plants and the production of metabolites.Their applications can be studies of physiology, genetics, biochemistry; procurement of pathogen-free plants, method for the conservation and exchange of germplasm as well as in vitro morphogenesis (Organogenesis or somatic embryogenesis) (Roca and Mroginski, 1991).
In vitro organogenesis in plants is a morphogenetic event which occurs due to structural changes and modifications in cellular organization, resulting in a unipolar organism; in in vitro tissue culture it can be direct (formation from an explant) or indirect (previous induction of the callus is required) (Sharry et al., 2015).In general, this process can be induced through the application of plant growth regulators (PGRs), the capacity of the tissue to respond to these factors during the culture, the type of culture medium and the organic additives (Sugiyama, 1999).
During the process of in vitro culture, the plants regenerated from non-meristematic cells or tissues often go through a phase of callus formation, usually exhibiting phenotypic and genetic variation (Ramulu and Dijkhuis, 1986;Kaeppler et al., 2000).These variations can affect the quality and quantity of the clones, as well as their application in the regeneration of genetically transformed tissues (Alatar et al., 2017).A frequent phenomenon found in in vitro culture is the spontaneous appearance of somaclonal variation (Larking and Scowcroft, 1981).
There are a number of reports in which the use of flow cytometry has enabled the analysis of changes in the stability of the clones; for example, in the species Vitis vinifera, Leal et al. (2006) reported the analysis of the ploidy level and Prado et al. (2010) detected somaclonal variants; moreover, in J. curcas, Franco et al. (2014) analyzed the clonal fidelity among genotypes, Rathore et al. (2014) genetic homogeneity and de Oliveira et al. (2013) performed the analysis of polyploidization.The aim of the present study was to develop a protocol of in vitro indirect organogenesis for J. curcas variety ALJC01, a high seed yield variety, in order to propagate plant material for future research and agronomic applications.Also, this is first report of in vitro culture of J. curcas in which the genetic stability of clones from one to eight subcultures was analyzed by flow cytometry.

Establishment of Jatropha curcas plant material
Seeds of J. curcas variety ALJC01 were collected randomly from a plantation in Sucila, Yucatan, Mexico.The seeds were surface sterilized with benzalkonium chloride at 0.5% and were then placed in Extran® at 1% for 15 min and left to dry on absorbent paper for 30 min.Subsequently, the seed coat was removed and the embryos obtained were disinfected in a laminar flow cabinet by immersion in a solution of Extran® at 5 % for 5 min.Afterwards they were transferred to ethanol at 70% for 1 min, followed by agitation in sodium hypochlorite at 30% for 15 min and by three final rinses in sterile distilled water.The disinfected embryos were transferred to magenta boxes with 40 mL of MS culture medium.All the embryos were incubated at a temperature of 23±2°C and relative humidity (RH) of 60% in a darkness growth chamber for 15 days, after which they were cultivated in a photoperiod of 16/8 hours (light/darkness); the light source was provided by LED lamps with a light intensity of 60 µmol -2 s -1 .

Induction of adventitious shoots
A 3 3 factorial design was constructed in which 27 treatments with 3 repetitions were obtained.The first factor was 6-(γ, γ-Dimethylallylamino) purine (2ip) at 96.00, 110.70 and 125.40 µM, the second factor was IAA at 1.10, 1.27 and 1.44 µM and the third factor was adenine sulfate (AdS) at 282.34, 325.78 and 369.21 µM; all the treatments were prepared with the 3/4 MS medium (Table 1).For the evaluation, the initial weight of the explant and the weight at 45 days were registered, and these values were used to calculate the average weight obtained during the growth period.The number of shoots formed at 45 days was also evaluated.

Development of adventitious shoots
A second 3 3 factorial design was used for root elongation, using PGR at 4.44, 6.66 and 8.88 µM, IAA at 1.0, 1.7 and 2.4 µM and the organic additive AdS at 543, 1086 and 814.5 µM; 27 treatments with three repetitions were established (Table 2).The experimental unit was a shoot with an initial length of 0.5 cm, which was measured after 45 days to determine its final length and to obtain total growth.

Rhizogenesis of shoots obtained from J. curcas
Shoots 2 cm in length were used to induce rhizogenesis.The experimental design was also a 3 2 factorial arrangement, with the factors being the ionic concentration of the MS basal medium at 25, 50 and 100% ionic strength and the IBA auxin at 14.7, 17.7 and 20.7 µM; the experimental design was constructed with and without phloroglucinol (Phl) (2.4 mgL -1 ).The variables evaluated after 30 days were the percentage of rooted shoots, length and number of developed roots.

Estimation of nuclear DNA content
For the isolation of nuclei, 100 mg of callus with leaf from J. curcas and 100 mg of leaf from Zea mays L. 'CE-777' (reference standard with 2C = 5.43 ; Lysak and Doležel 1998) were weighed and subsequently chopped with a razor blade in a Petri dish with 1000 µL of WPB solution (0.2 M Tris•HCl, 4 mM MgCl2•6H2O, 2 mM EDTA Na2.2H2O, 86 mM NaCl, 10 mM sodium metabisulfate, 1 % PVP-10, 1 % (v/v) Triton X-100, pH 7.5) (Loureiro et al., 2007).The nuclear sample was then filtered with a 40 µM nylon mesh and 50 µL of Propidium iodide (1mg/1mL) was added to stain nuclear DNA.After an incubation period of 15 min, the sample was analyzed in a BD Accuri C6® flow cytometer equipped with a blue and red laser, two light scatter detectors, and four fluorescence detectors with optical filters optimized for the detection of many popular fluorochromes.Data was obtained using BD Accuri™ C6 Plus software in the format of FCS 3.1 format; for each sub-cultured generation of J. curcas (until the eight subculture), three samples were analyzed in triplicate in three days.
The average fluorescence of 2C peaks of J. curcas ALJC01 variety and of Zea mays was obtained and the information was used to estimate the nuclear DNA content as follow: The estimated value of the nuclear DNA was converted to base pairs, considering 1 pg of DNA correspondent to 0.978x10 9 bp (Doležel et al., 2003).

Statistical Analyses
Statistical

Development of adventitious shoots
Shoot development was observed at 45 days of culture.In treatment T1 (4.44 µM of BAP, 1.0 µM of IAA and 543 µM of AdS), shoot elongation up to a length of 1.67±0.76cm was achieved (Table 2).Statistically significant differences were found regarding the number of leaves (P<0.05), with an average leaf development of 18.00±2.65leaves in treatment T1.Significant statistical differences were observed in the number of shoots (P<0.05); the treatment which presented the most significant shoot development was T2 with an average of 7.67±1.53shoots (Table 2).

Rhizogenesis of the developed shoots
Organogenesis of roots was obtained in J. curcas plantlets in culture media with the addition of IBA (Table 3).The treatments which presented the best response to root organogenesis were T10 (1/4 MS + 14.7µM of IBA), T15 (1/2 MS + 20.7 µM of IBA) and T16 (MS + 14.7µM of IBA); however, the highest number of roots generated was observed in treatment T15 (1/2 MS + 20.7 µM of IBA) with an average of 13.6±2.51roots and a length of 1.90±0.81cm (Figure 1).

Estimation of nuclear DNA content
The average fluorescence of 2C peaks obtained from the histograms of 1 to 8 subcultures was 3634.32±1519.02for J. curcas ALJC01 variety and 22922.1±10448.1 for Zea mays 'CE-777' (Figure 2).The average for the nuclear DNA content estimated for J. curcas, according to Carvalho et al. (2008), is 0.85 pg ±0.006; in this report, the nuclear DNA content in J. curcas for each one of the subcultures was found to range between 0.80±0.12and 1.07±0.23.Statistically significant differences were found for 3 rd and 4 th month of in vitro culture with a confidence level of 95.0% (Table 4).

Induction of adventitious shoots
The results obtained in this work are similar to those obtained by Gangwar et al. (2016) who achieved the formation of 25 shoots; it is pertinent to mention here that after 30 days it was possible to induce morphogenetic response in 70% from the cotyledonary leaf explants and at 45 days of culture the development of real leaves was observed (Figure 3).The results of the analysis of variance carried out (P<0.05) showed that the development of the shoots was due to the interaction of the principal effects 2ip, IAA and AdS.
In Figure 4a an average of 7 shoots can be observed with the interaction of the concentration of 125.40 µM of 2ip and 1.27 µM of IAA; in Figure 4b we can see that 125.40 µM of 2ip with 369.21 µM of AdS generated the same number of shoots, while in Figure 4c it was possible to observe the interaction of 1.27 µM of IAA and 369.21 µM of AdS which promoted the formation of 11 shoots.George et al. (2008) have indicated that AdS acts as a precursor of the natural synthesis of cytokinin or improves the natural biosynthesis of AdS and thus, the compounds produced could be more efficient in causing a physiological response than the cytokinins added to the culture medium.These benefits may often be noticed when they are associated with cytokinins, which may corroborate the interactions observed in Figure 4.It has also been demonstrated that AdS can serve as a precursor of the synthesis of zeatin (McGaw et al., 1984), which would indicate the possible effect on the induction of adventitious shoots in J. curcas.These results exceeded those obtained in the patent of Sreenivasachar et al. (2011) who obtained an average of 3 to 4 shoots.Our results also surpass those reported in the report of Varshney and Johnson (2010) and Geetaa and Sudheer (2011) who obtained the formation of 10 shoots per explant with the processes of indirect and direct organogenesis, respectively.
Recently, several authors (Liu et al., 2016;Verma and Verma, 2015;Liu et al., 2015) have reported the formation of 6 to 25 shoots per explant by means of direct and indirect organogenesis, using a diversity of J. curcas explants.

Development of adventitious shoots
AdS is an organic additive which has an effect on both the induction and elongation of the in vitro adventitious shoots of J. curcas; this may be due to the fact that the interaction of AdS with the cytokinins facilitates the growth and development of the shoots in in vitro culture (Nwankwo and Krikorian, 1983).This has been corroborated in a report by Shrivastava and Banerjee (2008) who were also able to achieve the induction and development of J. curcas shoots by combining AdS with BAP and other additives such as glutamine, L-arginine and citric acid.Figure 5 shows the elongated shoots of J. curcas at 45 days of culture with the growth of lateral shoots and development of leaves.(Doležel et al., 2003).Dewir et al. (2016) indicated that, in order to achieve a successful micropropagation system, optimal conditions for rooting and shoot development are required given that the search for a good number of healthy roots will allow the plantlets to establish in the soil and will promote normal growth and development.Although a few authors have reported that higher concentrations of IBA can induce higher levels of secondary metabolites and ethylene (Baker and Wetzstein, 1994;De Klerk, 2002), which could lead to the inhibition of the root formation process, the results obtained in this work showed that both the lowest (14.7 µM) and the highest (20.7 µM) IBA concentrations used, gave similar results.Even though the analysis of variance (p<0.05) did not indicate significant statistical differences, it seems that the ionic strength of the medium has a greater effect on the organogenic response of the roots, as can be seen in the Pareto diagram of Figure 6.It was interesting to note that, although the effect of Phl on root induction has been indicated (Daud et al., 2013) the results obtained with the MS medium treatments at different concentrations and the addition of different concentrations of IBA + Phl (Table 3) did not give efficient results.In fact, the highest number of roots obtained with Phl was in T5 (1/2 MS + 17.7 µM of IBA + 2.54 mg•L -1 of Phl) which generated 7.30 ± 2.30 roots, exceeded only by T15 with 13.6 ± 2.51 roots (Table 3).

Estimation of nuclear DNA content
The variation may have been due to the method of nuclear extraction, the type of fluorochrome and the cytometer used for the analysis of the sample (Doležel et al., 1992).In addition, the standard used for the measurement was Zea mays L. 'CE-777', which differs from the standard Raphanus sativus 'Saxa' used by Carvalho et al. (2008) in the estimation of the nuclear DNA content of J. curcas; Despite the fact that the propidium iodide adheres stoichiometrically to the DNA bases and can mark the nuclear DNA (Riccardi and Nicoletti, 2006), the rapid, active decondensation and condensation of the DNA (Belmont, 2003) could be another factor which may be influencing the results obtained, avoiding homogenous DNA staining in some nuclear populations.
For the multiplication of elite genotypes of J. curcas on a larger scale, it is important to confirm the genetic stability of the regenerated explants and to demonstrate the reliability of the regeneration systems, as demonstrated by Rathore et al. (2014) who evaluated J. curcas genotypes from tissues regenerated in vitro; moreover, detection of the ploidy level of DNA by flow cytometry was found to be a practical and rapid strategy for the selection of diploid, mixoploid and tetraploid plantlets induced in vitro from J. curcas meristems (de Oliveira et al., 2013).Also, indirect organogenesis is associated with higher levels of genetic instability.Soares et al. (2016) reported variation in the genetic stability over three generations of J. curcas subcultured by indirect organogenesis, which would suggest that this variation increases with each successive subculture.The analysis by flow cytometer of the nuclear DNA content in J. curcas plantlets, from one to eight subcultures, demonstrated that the genetic stability of the clones regenerated from J. curcas callus remains stable, indicating that the protocol reported in the present work is suitable for the propagation of J. curcas by indirect organogenesis, which will ensure that the clones can be maintained over eight generations without leading to changes in the nuclear DNA content.This protocol could be used for the genetic improvement of the species in the future, given that plant tissue culture and the techniques of molecular biology are biotechnological tools which can complement conventional reproduction, accelerate genetic improvement and satisfy the demand for the availability of uniform clones in large quantities (Mukherjee et al., 2011).

CONCLUSION
The leaf explant cells presented totipotency, allowing the induction of adventitious shoots with which it was possible to develop a protocol of morphogenesis via indirect organogenesis in J. curcas L. var.ALJC01.The protocol obtained can be used as an in vitro propagation technique for this valuable crop and for future studies on the regeneration of genetically transformed explants, which may be developed in this species.The genetic stability of the clones regenerated by indirect organogenesis was also assured in eight generations of J. curcas subcultured in vitro.

Figure 2 .
Figure 2. Histograms obtained in the flow cytometry of Jatropha curcas explants originating from 1 to 8 subcultures.a-h) first to eighth subculture in vitro.

Figure 3 .
Figure 3. Adventitious shoot induction of Jatropha curcas: a) explant with callogenesis; b) plantlet with 30 days of induction; and c) plantlet with adventitious shoot morphogenesis at 45 culture days, leaf development can be observed on shoots.

Figure 5 .
Figure 5. Adventitious shoot development of J. curcas during 45 culture days: a) principal shoot development with lateral shoot growth; b) lateral shoot development without apical dominance; and c) development of one shoot with apical dominance.

Figure 6 .
Figure 6.Standard Pareto diagram for number of formed roots.

Table 1 .
Average of weight gain and number of adventitious shoots in Jatropha curcas ALJC01 explants after 45

Table 2 .
Adventitious shoot development of Jatropha curcas.Information about total stem growth, number of leaves and number of adventitious shoots is also given.
a-g Similar letters correspond to treatments statistically equal according to the Tukey test p<0.05.All treatments were prepared with MS medium

Table 3 .
Rhizogenesis of the developed shoots in Jatropha curcas in vitro plants.Information about the percentage of explants with root development, number of roots and root length is provided.Similar letters correspond to treatments statistically equal according to the Tukey test p<0.05.

Table 4 .
Peak averages of histograms in explants of Zea mays and explants of Jatropha curcas; DNA content estimated from one to eight subcultures in vitro.