Methods to assess the viability of cryopreserved Jatropha curcas L. seed germplasm

Abstract

Jatropha curcas L. is a plant species with many potential applications, especially medicinal uses (hypoglycemic, anti-inflammatory, haemostatic, healing, anti-tumor). The objective of this study was to test germination in moist paper rolls for whole seeds and in vitro for excised embryonic axes, in an attempt to identify the best method to assess the quality of J. curcas seed germplasm, cryopreserved with different water contents. The experimental sample with a 6.2% moisture content (MC) was divided in subsamples which were hydrated and dehydrated for 0 (control), 4, 8, 11 and 24h. The initial germination percentages were 63% for whole seeds and 81% for excised embryonic axes. After exposure to liquid nitrogen (LN), germination percentages were 48% (whole seeds) and 57% (excised embryonic axes). There was no significant difference between germination percentages in embryonic excised from seeds subjected or not subjected to freezing, with different MC. In contrast, there was a reduction of the whole seed germination percentage when exposed to LN (contrast = 0.17, standard error = 0.04, t = 4.09, p = 0.001) and not for the hydration and dehydration treatments. The methodology based on in vitro cultures of the embryonic axis isolated from seeds stored in LN with distinct MC values was more efficient than the standard germination test to evaluate the viability of J. curcas seeds before and after LN storage.Keywords: seed, embryonic axis, cryopreservation, Jatropha curcas RESUMO: Métodos para avaliar a viabilidade de germoplasma semente criopreservado de Jatropha curcas L. Jatropha curcas L., é uma espécie com várias aplicações potenciais, principalmente para usos medicinais (hipoglicemina, anti-inflamatório, homeostático, cicatrizante, antitumoral). O objetivo deste estudo foi testar a germinação em rolo de papel para sementes inteiras e in vitro para eixos embrionários excisados visando identificar o melhor método para avaliar a qualidade de germoplasma semente de J. curcas, criopreservado com diferentes teores de água. A amostra experimental com 6,2% de teor de água foi dividida em subamostras que foram hidratadas e desidratadas por 0 (controle), 4, 8, 11 e 24 h. Os percentuais de germinação inicial foram de 63% para sementes inteiras e de 81% para eixos embrionários excisados. Após exposição ao nitrogênio líquido (NL) os percentuais de germinação foram de 48% (sementes inteiras) e 57% (eixos embrionários). Não houve diferença significativa entre os percentuais de germinação de eixos embrionários excisados de sementes com diferentes umidades e submetidas ou não ao congelamento. Em contraste, houve redução de percentuais de germinação das sementes inteiras expostas ao NL (contraste = 0.17, erro padrão = 0.04, t = 4.09, p = 0.001), mas não aos tratamentos de hidratação e desidratação. A metodologia baseada em cultura de eixos embrionários in vitro isolados de sementes armazenadas em NL, com distintos teores de água foi mais eficiente que a germinação padrão para avaliar a viabilidade de sementes J. curcas ...

Efficiency of Cryoprotectants for In Vitro and Ex Vitro Germination of Cryopreserved Jatropha curcas Seeds

“…Radhamani and Sivaraj (2013) emphasizes that the viability of orthodox seeds can be maintained by drying the seeds and storing at low temperatures, and jatropha shows orthodox seed storage behavior. Nonetheless, lipids present in the seed composition may make the seeds more sensitive to freezing in LN (Das Bhowmik et al, 2011;Salomão et al, 2016), as observed in this study. However, Chmielarz (2009) reports that oil seeds containing unsaturated fatty acids in its composition, such as jatropha, may have the negative effect of the transition phase during freezing attenuated.…”

“…However, a significant percentage of contamination in vitro caused by fungus was observed 7 DAS for T3, T4, T5, and T9, and 14 DAS for T1, T3, T4, T5, and T9. Similarly, Salomão et al (2016) reported significant reduction in percentages of jatropha seeds germination after exposure to LN, such as 48% (whole seeds) and 57% (excised embryonic axes), being this attributed to fungal contamination.…”

“…curcas seeds obtained after crosspollination were collected from twelve 4-year-old hybrids and stored in paper bags under laboratory conditions for three days. The mean values for length, width, 100 seed weight, and water content of these 4-d-old seeds before the cryopreservation process in LN were 1.70 cm, 1.87 cm, 71.38 g, and 6.9%, respectively, which is suitable for this species (Salomão et al, 2016). Seeds were washed in distilled water, followed by surface sterilization with 1% Alconox (Alconox, New York, NY) solution, and three successive distilled water rinses.…”

“…Although cryopreservation of plant material in the form of seeds and zygotic embryos is currently widely used (Kaviani, 2011;Nadarajan and Pritchard, 2014;Silva et al, 2012), even the best treatments in this study resulted in low percentage of germination in vitro 7 DAS (T1 and T6) and 14 DAS (T1 and T8), varying between 25% and 37.5%. This could be related to intrinsic traits of the seed, such as composition, moisture content, and tolerance to ultralow temperatures (Chmielarz, 2009;Das Bhowmik et al, 2011;Prada et al, 2015;Salomão et al, 2016).…”

“…In jatropha, in vitro seed cryopreservation protocols have been developed, but some problems related to this procedure have been reported, such as decrease in seed germination percentage (Salomão et al, 2016), low development of explants in vitro from cryopreserved seeds (Prada et al, 2015), and seedlings with reduced vigor and significant increase in abnormal plants (Silva et al, 2012). Therefore, the development of new cryopreservation protocols using alternative cryoprotectant combinations could improve the efficiency of this technique.…”