ABSTRACT
Abstract A cryopreservation protocol was developed for in vitro shoot tips of Garcinia hombroniana using the vitrification technique. Four critical steps in the technique were investigated, namely preculture, loading, dehydration with Plant Vitrification Solution 2 (PVS2), and unloading. Shoot tips precultured for 48 h gave significantly higher survival (75 %) compared to 24 h preculture (50 %) after cryopreservation. Treatment with 1 M glycerol plus 0.4 M sucrose as a loading solution gave higher survival (45.83 %) compared to the other treatments (0.4 M sucrose + 2 M glycerol; 0.4 M sucrose). Shoot tips dehydrated with PVS2 for 25 min gave the highest survival after immersion in liquid nitrogen. Stepwise PVS2 treatment for 15 min with 50 % PVS2 followed by 10 min with 100 % PVS2 solution improved survival of the shoot tips after cryopreservation (41.67 %). Murashige and Skoog medium with 0.4 M sucrose gave significantly higher survival (66.67 %) than MS with 1.2 M sucrose (25 %) as an unloading solution. Water content was shown to decrease throughout the whole vitrification steps from 6.83 ± 1.66 g g-1 dw for fresh shoot tips down to 2.93 ± 0.28 g g-1 dw after PVS2 treatment. Further study on each step including recovery medium is required to improve the survival. Nevertheless, the present study showed the potential of using the vitrification technique for cryopreservation of G. hombroniana. Rev. Biol. Trop. 66(3): 1314-1323. Epub 2018 September 01.
Resumen Se desarrolló un protocolo de crioconservación in vitro para ápices caulinares de Garcinia hombroniana mediante la técnica de vitrificación, con cuatro etapas críticas: precultivo, carga de crioprotector, deshidratación con solución de vitrificación vegetal 2 (PVS2), y descarga. Ápices precultivados por 48 h sobrevivieron más (75 %) que los de 24 h (50 %) después de la crioconservación. El tratamiento con glicerol 1 M más sacarosa 0.4 M como solución de carga permitió mayor sobrevivencia (45.83 %) que los otros tratamientos (sacarosa 0.4 M + glicerol 2 M; sacarosa 0.4 M). Los ápices deshidratados con PVS2 por 25 min registraron la mayor sobrevivencia tras inmersión en nitrógeno líquido. El tratamiento gradual por 15 min con solución de PVS2 al 50 %, seguido por 10 min al 100 %, mejoró la sobrevivencia de ápices tras la crioconservación (41.67 %). El medio Murashige-Skoog (MS) con sacarosa 0.4 M produjo una sobrevivencia significativamente mayor (66.67 %) que MS con sacarosa 1.2 M (25 %) como solución de descarga. El contenido de agua disminuyó a lo largo del proceso de vitrificación desde 6.83 ± 1.66 g g-1 peso seco, en ápices frescos, hasta 2.93 ± 0.28 g g-1 peso seco después del tratamiento con PVS2. Se requiere de más investigación sobre cada etapa, incluyendo el medio de recuperación, para mejorar la tasa de sobrevivencia. Sin embargo, este estudio muestra el potencial de la vitrificación para la crioconservación de G. hombroniana.
Subject(s)
Cryopreservation/instrumentation , Garcinia , Vitrification , Plants , Sucrose/therapeutic use , Glycerol/therapeutic use , Malaysia , Nitrogen/therapeutic useABSTRACT
The cytosolic mevalonate (MVA) pathway in Hevea brasiliensis latex is the conventionally accepted pathway which provides isopentenyl diphosphate (IPP) for cis-polyisoprene (rubber) biosynthesis. However, the plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway may be an alternative source of IPP since its more recent discovery in plants. Quantitative RT-PCR (qRT-PCR) expression profiles of genes from both pathways in latex showed that subcellular compartmentalization of IPP for cis-polyisoprene synthesis is related to the degree of plastidic carotenoid synthesis. From this, the occurrence of two schemes of IPP partitioning and utilization within one species is proposed whereby the supply of IPP for cis-polyisoprene from the MEP pathway is related to carotenoid production in latex. Subsequently, a set of latex unique gene transcripts was sequenced and assembled and they were then mapped to IPP-requiring pathways. Up to eight such pathways, including cis-polyisoprene biosynthesis, were identified. Our findings on pre- and post-IPP metabolic routes form an important aspect of a pathway knowledge-driven approach to enhancing cis-polyisoprene biosynthesis in transgenic rubber trees.
Subject(s)
Erythritol/analogs & derivatives , Gene Expression/genetics , Hevea/metabolism , Latex/analysis , Mevalonic Acid/metabolism , Rubber/metabolism , Sugar Phosphates/metabolism , Base Sequence , Carotenoids/metabolism , Erythritol/metabolism , Gene Library , Genes, Plant/genetics , Hevea/genetics , Molecular Sequence Data , RNA, Plant/genetics , Sequence Analysis, DNA , Terpenes/metabolism , TranscriptomeABSTRACT
Hevea brasiliensis is the most widely cultivated species for commercial production of natural rubber (cis-polyisoprene). In this study, 10,040 expressed sequence tags (ESTs) were generated from the latex of the rubber tree, which represents the cytoplasmic content of a single cell type, in order to analyse the latex transcription profile with emphasis on rubber biosynthesis-related genes. A total of 3,441 unique transcripts (UTs) were obtained after quality editing and assembly of EST sequences. Functional classification of UTs according to the Gene Ontology convention showed that 73.8% were related to genes of unknown function. Among highly expressed ESTs, a significant proportion encoded proteins related to rubber biosynthesis and stress or defence responses. Sequences encoding rubber particle membrane proteins (RPMPs) belonging to three protein families accounted for 12% of the ESTs. Characterization of these ESTs revealed nine RPMP variants (7.9-27 kDa) including the 14 kDa REF (rubber elongation factor) and 22 kDa SRPP (small rubber particle protein). The expression of multiple RPMP isoforms in latex was shown using antibodies against REF and SRPP. Both EST and quantitative reverse transcription-PCR (QRT-PCR) analyses demonstrated REF and SRPP to be the most abundant transcripts in latex. Besides rubber biosynthesis, comparative sequence analysis showed that the RPMPs are highly similar to sequences in the plant kingdom having stress-related functions. Implications of the RPMP function in cis-polyisoprene biosynthesis in the context of transcript abundance and differential gene expression are discussed.