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 ]]>
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 ]]>
in vitro did not germinate, and were 60% contaminated. The germination of the zygotic embryos was significantly higher in the ½ 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, 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 ]]>
in vitro plant recovery, when seeds are conserved in germplasm banks by low or cryotemperatures.
Actualmente, el ]]>
Jatropha ssp. se conserva como plantas enteras en las colecciones de campo. Bajo este método de almacenamiento, los recursos genéticos están expuestos a enfermedades, plagas y desastres naturales tales como el error humano, la sequía y las inclemencias del tiempo. Además, los bancos de germoplasma de campo son costosos de mantener y requieren bastante personal capacitado. Por lo tanto, el desarrollo de técnicas eficientes para asegurar su conservación segura así como su ]]>
Jatropha curcas después de descongeladas. En un primer experimento, se llevó a cabo un protocolo eficiente para la recuperación de plantas in vitro mediante el uso de embriones cigóticos o semillas con o sin testa. En un segundo experimento, las semillas disecadas, con o sin testa fueron expuestas a nitrógeno líquido y se evaluaron ]]>
J. curcas presentaron hasta un 99.6% de germinación después de la eliminación de la testa. Las semillas con la testa cultivadas in vitro no germinaron, y el 60% se contaminaron. ]]>
%) en comparación con el medio WPM (76.2%), pero desde los embriones zigóticos, las plántulas anormales alcanzaron más del 99%. Semillas con la testa inmersa en nitrógeno líquido mostraron un 60% de germinacion en cultivos ]]>
% de germinación, pero las plántulas mostraron un reducido vigor y un incremento significativo de plantas anormales. Semillas con testa cultivadas in vitro no germinaron, independientemente de la criopreservación o no. Este estudio reporta el primer éxito in vitro de una metodología de ]]>
Jatropha curcas, después de un tratamiento de criopreservación, que se recomienda como un procedimiento eficaz para la recuperación de plantas in vitro, cuando las semillas se conservan en bancos de germoplasma a bajas o crio-temperaturas.
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Palabras clave: Jatropha sp., conservación ex situ, criopreservación, recursos fitogenéticos, cultivo de tejidos vegetales, agroenergía, biodisel.
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).
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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 ]]>
% 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 ]]>
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 ]]>
Cryopreservation, i.e., the storage of biological material at ultra-low temperature, usually that of liquid nitrogen (-196°C, LN), is the only technique currently available to ensure the safe and cost-efficient long-term conservation of germplasm. ]]>
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 ]]>
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) (15o48’35” S - 43o18’28” W at 516m in elevation), and stored at ]]>
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 ]]>
% (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 ]]>
% (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 ]]>
& 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 ]]>
2s 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 ]]>
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 ]]>
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% ]]>
% (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 ]]>
All cultures were maintained in the same conditions used for in vitro germination procedure. In this ]]>
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 ]]>
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, ]]>
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 ]]>
Table 2).
A relatively high level was observed for the formation of abnormal seedlings, with atrophied cotyledonary leaves, low ]]>
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). ]]>
The initial moisture content of cryopreserved seeds was 9.8% with germination values of ]]>
% in the treatment where the seeds were stored and germinated without coat (Table 3). Seeds cultured in vitro with coat did not 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 ]]>
% 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 ]]>
%, 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.
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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 ]]>
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% ]]>
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).
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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 ]]>
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 ]]>
Cryopreservation of plant materials in the form of whole seeds, excised embryos, reproductive parts and ]]>
et al. 2009). Thus, the result would be the improved maintenance of ]]>
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 ]]>
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 ]]>
% 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 ]]>
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 ]]>
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 ]]>
% 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 ]]>
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 ]]>
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) ]]>
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 ]]>
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 ]]>
Jatropha species and accessions is currently under investigation.
Acknowledgments
The authors thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brasília, DF, Brazil, for financial support and ]]>
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*Correspondencia a:Rafael de C. Silva: Post-Graduate Program in Biotechnology, Federal University of Amazonas, 69077-000 Manaus, AM, Brazil; carvalho_fael@yahoo.com.br Julcéia Camillo: Post-Graduate Program in Agronomy, University of Brasília, 70910-900 Brasília, DF, Brazil; julceia@gmail.com Jonny E. Scherwinski-Pereira: Embrapa Genetic Resources and Biotechnology, Plant Tissue Culture Laboratory, P.O. Box 02372, Brasília, DF, Brazil; jonny@cenargen.embrapa.br
1. Post-Graduate Program in Biotechnology, Federal University of Amazonas, 69077-000 Manaus, AM, Brazil; carvalho_fael@yahoo.com.br 2. Post-Graduate Program in Agronomy, University of Brasília, 70910-900 Brasília, DF, Brazil; julceia@gmail.com 3. Embrapa Genetic Resources and Biotechnology, Plant Tissue Culture Laboratory, P.O. Box 02372, Brasília, DF, Brazil; jonny@cenargen.embrapa.br
Received 25-X-2010. Corrected 10-V-2011. Accepted 09-VI-2011.
