A method for seedling recovery in Jatropha curcas after cryogenic exposure of the seeds

Actually, the germplasm of Jatropha spp. is conserved as whole plants in field collections. Under this storage method, the genetic resources are exposed to disease, pest and natural hazards such as human error, drought and weather damage. Besides, field genebanks are costly to maintain and with important requirements of trained personnel. Thus, the development of efficient techniques to ensure its safe conservation and regeneration is therefore of paramount importance. In this work we describe a method for Jatropha curcas seeds cryoexposure and seedling recovery after thawed. In a first experiment, an efficient protocol for in vitro plant recovery was carried out using zygotic embryo or seeds with or without coat. In a second experiment, desiccated seeds with or without coat were exposed to liquid nitrogen and evaluated after cryoexposure. Germination percentages were variable among treatments, and seeds demonstrated tolerance to liquid nitrogen exposure under certain conditions. Seeds of J. curcas presented up to 99.6% germination after seed coat removal. Seeds with coat cultured in vitro did not germinate, and were 60% contaminated. The germination of the zygotic embryos was significantly higher in the 1⁄2 MS medium (93.1%) than in WPM medium (76.2%), but from zygotic embryo, abnormal seedlings reached up to 99%. Seeds with coat exposed to liquid nitrogen showed 60% germination in culture after coat removal with good plant growth, and seeds cryopreserved without coat presented 82% germination, but seedlings showed a reduced vigor and a significant increase in abnormal plants. Seeds cultured in vitro with coat did not germinate, independently of cryoexposure or not. This study reports the first successful in vitro seedling recovery methodology for Jatropha curcas seeds, after a cryopreservation treatment, and is recommended as an efficient procedure for in vitro plant recovery, when seeds are conserved in germplasm banks by low or cryotemperatures. Rev. Biol. Trop. 60 (1): 473-482. Epub 2012 March 01.

Recently, biodiesel has become more attractive because of its environmental benefits and the fact that it is made from renewable resources.Considerable research has been done on vegetable oils as diesel fuel.Jatropha curcas (Linnaeus) is a hardy perennial shrub or tree thought to be native from Central America and possibly from Brazil (Carvalho et al. 2008).Jatropha is a large genus comprising more than 170 species that is widespread throughout the tropical regions of the word (Deore & Johnson 2008).Most of these species are ornamental, except for J. curcas and J. glandulifera, which are oil-yielding species (Swarup 2004).
In fact, there is a growing interest in the cultivation of J. curcas for the production of oil as a fossil fuel substitute.The seeds of J. curcas contain at least 30-40% oil with a fatty acid pattern similar to that of edible oils (Gubitz et al. 1999).Jatropha oil contains linoleic acid and oleic acid, which together account for up to 80% of the oil composition.Palmitic acid and stearic acid are other fatty acids present in this oil (Deore & Johnson 2008).The fact that the oil of J. curcas cannot be used for nutritional purpose without detoxification makes its use as an energy source for fuel production very attractive.Besides, this euphorbia is a droughtresistant plant which grows on wasteland and could easily be cultivated by low income farmers.Also, like all trees, it fixes atmospheric carbon (Openshaw 2000).
However, the full potential of Jatropha is far from being realized.The growth and management is poorly documented and it lacks an improved germplasm for high yielding.While J. curcas germplasm is being harvested all over the world with the purpose of crop improvement, little is known about the germination and storage behavior of Jatropha seeds in order to improve the breeding programmes (Carvalho et al. 2008).Actually, the germplasm of Jatropha spp. is conserved as whole plants in field collection.With this storage method, the genetic resources are exposed to disease, pest and natural hazards such as human error, drought, and weather damage.In addition, field genebanks are costly to maintain and trained personnel requirements are very important (Engelmann 1991).Thus, the development of efficient techniques to ensure its safe conservation is therefore of paramount importance.
Cryopreservation, i.e., the storage of biological material at ultra-low temperature, usually that of liquid nitrogen LN), is the only technique currently available to ensure the safe and cost-efficient long-term conservation of germplasm.At this temperature, all cellular divisions and metabolic processes are decreased at minimum limits and, the plant material can thus be stored for a theoretically unlimited period of time (Walters et al. 2004).Moreover, cultures are stored in a small volume, protected from contamination, and require a very limited maintenance (Engelmann 2004).
Although research on the development of conservation and cryopreservation techniques have been conducted with seeds of numerous plant species (Walters et al 2004, Chmielarz 2009 a,b), up to now, there is no report in a methodology for seedling regeneration of Jatropha species after treatments of cryopreservation.This paper presents the first successful report on seedlings recovery after cryopreservation of Jatropha curcas seeds.

MATERIALS AND METHODS
Mature seeds of Jatropha curcas were obtained from growers of Janaúba City (Minas Gerais, Brazil) (15 o 48'35" S -43 o 18'28" W at 516m in elevation), and stored at temperature of 27±2°C until use.Seeds were weighted and oven-dried at 105°C for 24h to constant weight.The initial seed moisture content was estimated using three replicates of 10 seeds.

In vitro germination procedures:
In order to develop an efficient protocol for in vitro plant recovery after cryopreservation process, treatments were carried out as the following procedure: after being washed in running tap water for five min, the seeds were surface sterilized by soaking in 70% (v/v) ethanol for three min, and 2.5% (w/v) sodium hypochlorite for 20 min.Seeds were rinsed three times with sterile distilled.For in vitro germination, three treatments including seeds with (1) or without (2) coat and zygotic embryos (3) were evaluated.For seeds without coat, before the inoculation on culture media, a second superficial sterilization was realized in 70% (v/v) ethanol for one min, and 1.25% (w/v) sodium hypochlorite for 15min, following by rinsing three times in sterile distilled water.Zygotic embryo isolation was performed under sterile conditions, in a laminar flow hood, with the help of surgical blades and tweezers.
The explants were placed into test tubes (25x150mm) on MS (Murashige & Skoog 1962) with the macro-and micronutrients at half-strength (½ MS), and WPM (Wood Plant Medium, Lloyd & McCown 1980) medium, without plant growth regulation.All media contained 2% (w/v) sucrose, 0.6% agar and 0.03% (w/v) activated charcoal.The media were adjusted to pH 5.8 before being autoclaved at 121°C for 15min.The explants were cultured under 12h light/12h darkness photoperiod (50μmol/m 2 s light intensity provided by daylight white fluorescent tubes) and 26±2°C.Seed germination in vitro, development of seedlings (plant height, number of roots) and culture contamination were determined weekly for 21 days.
This experiment was conducted as completely randomized design with five replications and 20 seeds/replicate.

Cryoexposure of seeds:
A lot composed of 300 seeds was divided in five treatments (60 seeds each), being the two first considered as control (no exposure to LN).Before cryopreservation, seeds were hermetically sealed in laminated aluminum foil packets (15 seeds per packet), and placed in cryotanks to be exposed directly into LN.After 24h of storage in LN, seeds were thawed at room temperature (22-25°C) for 24h and germinated.
The germination test-control and cryopreserved seeds -were carried out on ½ MS medium, with 0.03% (w/v) activated charcoal, as the following treatments: 1) whole seeds (with the coat) sterilized in 70% (v/v) ethanol for 3min and 2.5% (w/v) sodium hypochlorite for 20min; 2) whole seeds (without the coat), the same procedure of treatment one was followed, but after seed coat was removed, a new sterilization was made to the seed endosperm with 70% (v/v) ethanol and 1.25% (w/v) sodium hypochlorite for 15min, followed by rising three times in sterile distilled water (control); 3) cryoexposure of seeds after seed coat removal, following the sterilization of endosperm after thawed; 4) cryoexposure of seeds with coat, following the superficial sterilization of the seed after thawed, and 5) cryoexposure of seeds with coat, following by tegument removed and superficial sterilization of the tegument after thawed.
All cultures were maintained in the same conditions used for in vitro germination procedure.In this experiment, fifteen seeds were used per treatment, and the experiment was replicated four times.
The following parameters were assessed: germination (%), shoot height (cm), fresh (FW) and dry (DW) matter weight of aerial, roots and whole plants (g).Germination was determined for all treatments every seven days for 21 days, and it was considered when the radicle protruded at least 3mm.Seedling emergence was estimated to observe abnormal seedlings formation.The plant height was considered as the distance between the medium surface and the insertion of the last visible leaf sheath.After 21 days the roots and aerial parts of five plants per treatment, selected at random, were used to measure fresh and dry matter weight (at 65°C, until constant weight).After a month in culture, the remaining seedlings were transferred to the greenhouse into pots with commercial substrate (Plantmax®), and observed over a period of up to three months for any phenotypic variation.
The experiment was repeated twice, and all data were subjected to analyses of variance (ANOVA) and comparisons of means were made with Tukey's multiple comparison Test at 5%.

RESULTS
Seeds of J. curcas after coat removal presented 99.6% and 98.3% germination at the end of 21 days of in vitro culture, on ½ MS and WPM media, respectively.Significant differences were observed between seeds and zygotic embryos.Zygotic embryos present lower germination in the WPM, compared with those of the ½ MS medium, with germination percentages of 76.2% and 93.1%, respectively.Seeds with coat on the ½ MS and WPM media did not germinate, and contamination reached 62.7% and 67.3% in both media, respectively, demonstrating a need for larger studies to improve the sterilization method (Table 1).
Seeds without coat germinated on WPM resulted in higher growth of the seedlings, with an average height of 7.7cm after 21 days of in vitro growth.However, seeds in the same conditions, germinated on ½ MS medium, showed lower growth, presenting an average height of 5.6cm after 21 days.In relation to the number of roots formed after 21 days of seedling growth, no significant differences were observed between the types of explants used or between the culture media tested (Table 2).
A relatively high level was observed for the formation of abnormal seedlings, with atrophied cotyledonary leaves, low vigor and delayed root development (Fig. 1).Seeds cultured in WPM and ½ MS medium after coat removal formed on average 20.8% and 33.9%, of abnormal seedlings respectively.For zygotic embryos, the percentage of abnormal seedlings reached up to 99.6% (Table 2).However, with the exception of seedlings originating from zygotic embryos which did not revert the characteristics of abnormality, all the seedlings of the other treatments, germinated from seeds returned to normal development after acclimatization.
The initial moisture content of cryopreserved seeds was 9.8% with germination values of 82.4% in the treatment where the seeds were stored and germinated without coat (Table 3).Seeds cultured in vitro with coat did not  Means following by different letters, small letters within a column, and capital letters along rows, within each variable evaluated, differ for the Tukey's test (p≤0.05).
(+) teg: seeds with coat; (-) teg: seeds without coat; ZE: zygotic embryo.germinate, and there were no significant differences between these and the control (-LN).However, seeds placed to germinate after removal of the coat (control) presented germination around 91%.In these plants, after 21 days of germination the occurrence of 10% of abnormal plants was observed, with low vigor, atrophied cotyledonary leaves and delayed root development.The percentage of contaminated seeds submitted or not to cryogenic conditions were 77.3% and 62.9%, respectively for those in which the coat was left intact at the moment of germination, showing that the sterilization for seeds with coat was not efficient.On the other hand, it was observed that the percentage of contaminated seeds immersed or not in liquid nitrogen, after removal of the coat, was less than 2% (Table 3).
Seeds cryopreserved with coat and then cultured in vitro without coat, presented around 60% germination after 21 days of in vitro culture.This is significantly lower than that observed for the control treatment and for seeds cryopreserved without the coat (82%, no statistical difference in relation to the control treatment).However, as described previously, contamination was 77.3% in the seeds cultivated with coat, reaffirming the results for the control treatment, a factor which was eliminated with the removal of the coat at the moment of germination in vitro.
In relation to the height of the seedlings, the seeds exposed to liquid nitrogen with coat and germinated in vitro without coat presented vigorous seedlings, with an average height of 7.3 cm at the end of the 21 days of in vitro culture, and did not differ statistically in relation to the control (Table 3).However, in the seeds submitted to liquid nitrogen without coat, even though these presented high levels of germination, reduced vigor was observed, and in this case the plants presented an average height of 3.9cm (Table 3).Such plants produced also a lower number of roots (Table 4).As for the number of leaves, no significant differences were observed between the treatments, with around two leaves per plant after 21 days of cultivation.All the abnormal plants from zygotic embryo and from seeds exposed without coat, when acclimatized, were unable to withstand the acclimatization and died, while those with normal germination and development or those exposed to LN with tegument and germinated without coat survived in 100% in the greenhouse.Such plants demonstrated phenotypically normal growth after two months of acclimatization (Fig. 1), although freezing the seeds of J. curcas without the coat resulted in a significant increase in abnormal plants morphologically (31.7%)(Table 4).
In general, seeds exposed to liquid nitrogen present a lower accumulation of fresh and dry mass, particularly those exposed without coat (Table 5).Thus, seeds with coat submitted to liquid nitrogen and cultivated without coat in culture medium resulted in seedlings with a fresh and dry weight which was lower than that of the control treatment, on average 1.3g and 0.12g, respectively.However, the fresh and dry weight of the root did not differ significantly from the control.The seedlings regenerated from the seeds exposed to the liquid nitrogen without coat presented lower vigor than the other treatments.However, the dry and fresh weight of the root did not differ significantly from the cryopreserved seeds with coat, presenting on average 0.1g and 0.01g respectively.

DISCUSSION
Plant genetic resources or germplasm describes the total genetic diversity of cultivated species and their wild relatives (Ford-Lloyd & Jackson 1991), and conservation of germplasm is the most fundamental aspect of biological conservation.Plant germplasm can be derived from a variety of sources, which will in turn determine the method of its conservation.Seed storage is the most common method used to conserve plant germplasm because of the convenience and relatively low costs involved.This strategy, however, cannot be applied to all plant species as the post-harvest behaviour of their seeds, which determines the most suitable method of conservation, differs.
Cryopreservation of plant materials in the form of whole seeds, excised embryos, reproductive parts and vegetative tissues is gaining momentum as a method for the conservation of germplasm.Cryopreservation refers to preservation at -196°C, the temperature of liquid nitrogen (LN).The basic concept of LN preservation is that, at this temperature, all metabolic processes in seeds are essentially reduced.Consequently, all sources of seed deterioration that are metabolically related are greatly reduced or stopped, thus providing 'indefinite' preservation.Storage in LN could prevent or significantly reduce such viability loss (Camillo et al. 2009).Thus, the result would be the improved maintenance of valuable genetic resources.
Given the variety and specific nature of factors affecting recovery after cryopreservation, specific in vitro conditions need to be optimized before cryopreservation can be attempted.Thus, the optimum culture medium for in vitro regeneration must also be ascertained.In this work, the initial moisture content of cryopreserved seeds was 9.8% with germination values of 82.4% in the treatment where the seeds were stored and germinated without coat.Seeds germinated without coat into MS and WPM media did not presented significant differences on the germination percentage and just for zygotic embryo the germination was significantly higher in the ½ MS medium than in the WPM medium, despite abnormal germination reached between 97% to 99% of zygotic embryo in culture, independently of media.It is interesting to note that MS medium has been successfully used in the in vitro culture of various tropical woody plant species, such as Caryocar brasiliense Camb.(Landa et al. 2000), Lychnophora pinaster Mart.(Souza et al. 2003) and Byrsonima intermedia A. Juss.(Nogueira et al. 2004).Experiments with mature, immature and dry seeds of J. curcas also revealed that MS medium was efficient to sustain germination and continuous growth of plants under in vitro conditions (Nunes et al. 2008).On the other hand, seeds cultured in vitro with coat did not germinate, and there were no significant differences between these and the control (-LN).These results can probably be attributed to the presence of dormancy in the seeds, as observed by Añez et al. (2005) who studied the germination behavior of seeds of J. elliptica, and observed that these seeds present physical dormancy caused by the impermeability of the coat, requiring a scarification treatment.It is particularly important to observe also that the occurrence of contamination was 77% in the seeds cultivated with coat, reaffirming the results for the control treatment, a factor which was eliminated with the removal of the coat at the moment of germination in vitro.However, for successful cryopreservation, it is necessary to avoid lethal intracellular freezing, which occur during rapid cooling in LN.In the present study, it was interesting to note that as well as the seeds cryopreserved with coat when seeds were cryopreserved without coat there was germination, but seedling presented reduced vigor, and in this case the plants presented an average height and number of roots per plant lower than those cryopreserved with coat.According to Normah et al. (1994) this decline in plant growth and number of roots was probably due to dormancy induced during dry storage of the seeds on embryo axis, as observed with hazelnut (Corylus avellana), besides a reduction in metabolism caused by exposure of the plant materials to subzero temperatures (Ellis & Roberts 1981, Hor et al. 2005).Furthermore, seeds without coat directly frozen in liquid nitrogen resulted in a significant increase in abnormal plants.This fact can be attributed to injury in the seed tissues, caused by exposure to extreme temperatures.
These results confirm the importance of cryopreserving seeds of J. curcas with the coat intact.Almeida et al. (2002) reports that seeds with high oil content, due to their chemical composition, are more susceptible to injury and physical damage caused by the exposure to low temperatures.According to Crane et al. (2003) and Volk et al. (2006), low temperatures crystallize lipids during seed storage and water interactions with crystallized storage lipids can be lethal to seeds.Thus, the seed coat maintenance could prevent this physic-chemical reaction preserving the viability of the seeds, although the reason why water contacting crystallized triacylglycerols is lethal to seeds is not known.
This study confirmed that J. Curcas is an orthodox seed and whole seeds can be cryopreserved under certain conditions.To our knowledge this is the first report of cryopreservation of seeds of J. curcas, which can contribute to the development of an efficient method for long-term conservation of genetic resources of this species.The procedure described for seedling recovery of cryopreserved seeds of Jatropha curcas is relatively simple and reliable whereby cryopreserved seeds germinated and seedlings developed into normal and healthy plants.Although this investigation has been a screening test to establish if J. curcas seeds are tolerant to liquid nitrogen when exposure is for a short time, the results obtained indicate that these seeds can withstand LN exposure without detrimental effects and cryopreservation would be another option to their germplasm conservation.Although under in vitro condition the germination percentages are high when seeds are deprived of coat, the presence of coat prior to LN exposure was essential to allow proper germination of cryopreserved seeds.In the present study, with the exception of seedlings originating from zygotic embryos which did not revert the characteristics of abnormality, all the seedlings of the other treatments returned to normal development after acclimatization.The application of this protocol for seedling recovery to other Jatropha species and accessions is currently under investigation.

TABLE 2
In vitro plant height (cm), number of roots (per plant) and abnormal plants (%) from cryopreserved (24h)

TABLE 5
Fresh (FW) and dry matter (DW) weight of aerial parts, roots and whole plant from cryopreserved (24h) Means followed by different letter within a column differ for the Tukey's test (p≤0.05).(+) coat: seeds with coat; (-) coat: seeds without coat; (+) LN: exposure to liquid nitrogen; (-) LN: non exposure to liquid nitrogen.