Somatic Embryogenesis In Oil Palm Research Articles

The protein model of somatic embryogenesis genes of oil palm (Elaeis guineensis Jacq.) using Phyre2 and Swiss-model

Somatic embryogenesis (SEs) is the capacity of plant-generated embryo structures to form a somatic cell. SEs in oil palm is one of the techniques used to produce clones with superior characteristics. It can maximize the oil palm crop’s productivity, whose demand increases every year. The identification of the structure of proteins can optimize the efficiency of the selection of the embryogenic culture. The somatic embryogenesis genes were collected from GeneBank at the NCBI. This study successfully describes the protein modeling of tree somatic embryogenesis genes in oil palms by Phyre2 and the Swiss model software. The ENOD93, HOX1 and RIk-OPff with the varied protein prediction the protein model of ENOD93 was detected with confidence of 81.5% by Phyre2, sequence identity of 76.6% with a range of 3-96 amino acids, and sequence similarity of 0.63 by Swiss-model. The sequence similarity of HOX1 was detected at 100% with a confidence level of 99.7% by the Swiss model. The protein model of RIk-OPff was predicted with 100% confidence in the range of 30–536 amino acids, and only in these genes were the trans-membrane helices.

Somatic embryogenesis in oil palm from immature leaves with emphasis on leaf position, sequential callus re-collection, use of temporary immersion system, and assessment of genetic and epigenetic fidelity of the resulting clones

Somatic embryogenesis in oil palm from immature leaves with emphasis on leaf position, sequential callus re-collection, use of temporary immersion system, and assessment of genetic and epigenetic fidelity of the resulting clones

Caffeate may play an important role in the somatic embryogenesis of oil palm (Elaeis guineensis Jacq.)

Somatic embryogenesis (SE) is a key step in plant regeneration and propagation, but the mechanisms underlying SE in oil palm still stay unclear. Integrated analysis of metabolome and transcriptome data showed that 13 metabolites are specifically accumulated in somatic embryo, of which caffeate was 16.6-fold (p = 0.0002) higher than in callus. Moreover, caffeate was significantly correlated with phytohormone signaling genes including ethylene receptor (ETR2-2), auxin efflux carrier component 3a (PIN3A), auxin response factor (ARF15-2), and several SE transcription factors (TFs) including WUSCHEL-related homeobox (WOX), VIVIPAROUS1(VP1), BABY BOOM2 (BBM2). Furthermore, staining and microscopic observation showed that phenolic acids highly accumulated in the center of newly formed callus clusters and lignin is predominant in somatic embryo than in callus, indicated that caffeate (the major phenolic acids in glycosylated form and the precursor of lignin biosynthesis) may play an important role in SE. Our results provide a better understanding of the SE mechanism in oil palm.

Genome-Wide Identification of WUSCHEL-Related Homeobox Gene Family and their Expression Analysis During Somatic Embryogenesis in Oil Palm (Elaeis guineensis)

Genome-Wide Identification of WUSCHEL-Related Homeobox Gene Family and their Expression Analysis During Somatic Embryogenesis in Oil Palm (Elaeis guineensis)

Effect of Forchlorfenuron on Somatic Embryo Proliferation and Plantlet Regeneration in Oil Palm SUP-PSU1

In vitro propagation of palm oil is still an obstacle for mass propagation due to the low plant regeneration frequency. The objective of this study was to investigate the effect of N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU) on a somatic embryo (SE) proliferation and plant regeneration of oil palm SUP-PSU1. At an approximate size of 5 mm, SE from chopping with a sharp razor blade at 100 times was cultured on oil palm culture medium (OPCM) with 0.1 mg/L dicamba and different concentrations CPPU. The results showed that 0.3 mg/L CPPU gave the highest SE proliferation at 100 % and the number of SEs at 5.16 embryos/tube after 4 weeks of culture. SE was transferred to Murashige and Skoog (MS) medium supplemented with 0.2M sorbitol, a secondary somatic embryo (SSE), which was induced at 66.67 % with 21.2 embryos/tube after 8 weeks. Furthermore, cultured SSE on PGR-free MS medium for 8 weeks gave the highest germination rate at 65 %, with the number of shoots at 2.29 shoots. Thus, this study provides new information for improving the plantlet regeneration system through somatic embryogenesis in oil palm.

Early nodulin 93 protein gene: essential for induction of somatic embryogenesis in oil palm.

Transcript profiling during the early induction phase of oil palm tissue culture and RNAi studies in a model somatic embryogenesis system showed that EgENOD93 expression is essential for somatic embryogenesis. Micropropagation of oil palm through tissue culture is vital for the generation of superior and uniform elite planting materials. Studies were carried out to identify genes to distinguish between leaf explants with the potential to develop into embryogenic or non-embryogenic callus. Oil palm cDNA microarrays were co-hybridized with cDNA probes of reference tissue, separately with embryo forming (media T527) and non-embryo (media T694) forming leaf explants sampled at Day 7, Day 14 and Day 21. Analysis of the normalized datasets has identified 77, 115 and 127 significantly differentially expressed genes at Day 7, Day 14, and Day 21, respectively. An early nodulin 93 protein gene (ENOD93), was highly expressed at Day 7, Day 14, and Day 21 and in callus (media T527), as assessed by RT-qPCR. Validation of EgENOD93 across tissue culture lines of different genetic background and media composition showed the potential of this gene as an embryogenic marker. In situ RNA hybridization and functional characterization in Medicago truncatula provided additional evidence that ENOD93 is essential for somatic embryogenesis. This study supports the suitability of EgENOD93 as a marker to predict the potential of leaf explants to produce embryogenic callus. Crosstalk among stresses, auxin, and Nod-factor like signalling molecules likely induces the expression of EgENOD93 for embryogenic callus formation.

Oil Palm Phytochrome-Interacting Factor4 (PIF4) Gene is Conserved and Highly Expressed During Somatic Embryogenesis

Oil palm is used in food, fuel and cosmetic industries. Tissue culture is the best way to propagate oil palm; unfortunately the somatic embryogenesis during tissue culture takes long time. The molecular mechanism of somatic embryogenesis in oil palm remains unknown. Recent research reported that auxin plays an important role in early and post-embryogenic plant. PHYTOCHROME-INTERACTING FACTOR4 (PIF4) regulates levels of auxin and the expression of key auxin biosynthesis genes. Our research aims to characterize oil palm PIF4 gene. Thus, we cloned EgPIF4, analyzed the domain using bioinformatic and examined the expression of EgPIF4 during somatic embryogenesis at different tissue including callus and somatic embryo stages; globular, torpedo, cotyledon, and plantlet stage using real-time PCR method. The result showed that EgPIF4 gene comprised 1,737 bp with 9 exons, which encode 578 amino acid residuals. It contains a conserved domain called basic helix-loop-helix domain. EgPIF4 has high level of expression at somatic embryogenetic stage specifically globular and torpedo stage suggested that EgPIF4 plays an important role during somatic embryogenesis. The future characterization of EgPIF4 function in oil palm will help to understand somatic embryogenesis process and facilitate the improvement of the oil palm tissue culture.

Oil Palm Phytochrome-Interacting Factor4 (PIF4) Gene is Conserved and Highly Expressed During Somatic Embryogenesis

Oil palm is used in food, fuel and cosmetic industries. Tissue culture is the best way to propagate oil palm; unfortunately the somatic embryogenesis during tissue culture takes long time. The molecular mechanism of somatic embryogenesis in oil palm remains unknown. Recent research reported that auxin plays an important role in early and post-embryogenic plant. PHYTOCHROME-INTERACTING FACTOR4 (PIF4) regulates levels of auxin and the expression of key auxin biosynthesis genes. Our research aims to characterize oil palm PIF4 gene. Thus, we cloned EgPIF4, analyzed the domain using bioinformatic and examined the expression of EgPIF4 during somatic embryogenesis at different tissue including callus and somatic embryo stages; globular, torpedo, cotyledon, and plantlet stage using real-time PCR method. The result showed that EgPIF4 gene comprised 1,737 bp with 9 exons, which encode 578 amino acid residuals. It contains a conserved domain called basic helix-loop-helix domain. EgPIF4 has high level of expression at somatic embryogenetic stage specifically globular and torpedo stage suggested that EgPIF4 plays an important role during somatic embryogenesis. The future characterization of EgPIF4 function in oil palm will help to understand somatic embryogenesis process and facilitate the improvement of the oil palm tissue culture.

HISTOLOGICAL ANALYSIS OF INDIRECT SOMATIC EMBRYOGENESIS INDUCED FROM ROOT EXPLATS OF OIL PALM (Elaeis guineensis Jacq)

ABSTRACT Oil palm is economically important as a crop with high oil production. Indirect somatic embryogenesis in oil palm requires a long time for callus induction and plant formation, and it is important to study the embryogenic potential of calli and the mechanisms of somatic embryogenesis. The aim of this study was to test different growth regulators and spermine in induction of embryogenic calli in root explants of oil palm (Elaeis guineensis Jacq). Apex root explants of approximately 0.5 cm were isolated from plants cultivated in vitro and inoculated in Y3 culture medium in the following treatments: A - without growth regulators; B - 1 mg.L-1 picloram (4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid); C - 1 mg.L-1 picloram and 2 mg.L-1 2ip (2-isopentenyladenine); D - 2 mg.L-1 2ip; E - 1 mg.L-1 picloram and 2 mg.L-1 BAP (6-benzylaminopurine); F - 2 mg.L-1 BAP; and G - 14.5 mg.L-1 spermine. After six months of culturing, the calli induced in the treatments were analyzed by light microscopy. The calli induced in the treatments with 1 mg.L-1picloram (B) and treatment with 14.5 mg.L-1spermine (G) exhibited embryogenic characteristics, small and isodiametric cells, forming agglomerates, besides a large amount of starch. Calli of the best treatment (Y3 com 1 mg.L-1 de picloram) were inoculated in Y3 culture medium without addition of growth regulators. After eight months, calli were once more analyzed under light microscopy. All the treatments showed callus formation, except for treatments D and A. Calli of treatment B exhibited cells with embryogenic characteristics that developed somatic embryos.

Genotype-dependent changes of gene expression during somatic embryogenesis in oil palm hybrids (Elaeis oleifera x E. guineensis).

To understand the molecular processes triggered during the different steps of somatic embryogenesis (SE) in oil palm, the expression of 19 genes associated to SE identified in proteomic and transcriptomic studies was investigated by qRT-PCR. To evaluate the differential expression of these genes, two interspecific hybrid genotypes (Elaeis oleifera x Elaeis guineensis) contrasting for the acquisition of embryogenic competence were used. Aclorophyllated leaves of both hybrids, one responsive (B351733) and the other non-responsive (B352933) to SE were submitted to callus induction and collected at different time points: 0 (before induction), 14, 30, 90 and 150 days of callus induction (doi). The results obtained showed that all evaluated genes were downregulated at 14 doi in the responsive genotype when compared to the non-responsive. It was also possible to observe that most of the genes changed their expression behavior at 30 doi and were upregulated thereafter until 150 doi, with the exception of the pathogenesis-related PRB1-3-like (PRB1-3) gene, which did not show differential expression at 30 doi and was downregulated at 90 and 150 doi when compared to the non-responsive hybrid. These results indicate that 30 doi is a turning point in gene expression, probably associated to embryogenic competence acquisition. We also show that the expression behavior of the responsive genotype is more stable than that of the non-responsive when the different induction time points are compared to 0 doi (before induction). Moreover, the results obtained in this study corroborate our hypothesis that the regulation of genes involved in the control of oxidative stress and energy metabolism are crucial for the acquisition of embryogenic competence in oil palm.

Gene Expression Analysis of Somatic Embryogenesis in Oil Palm

Multiplication of oil palm through somatic embryogenesis is hampered by low callogenesis and embryogenesis rates. Molecular marker based on RNA (transcriptomes analysis) is considered as one of the most e?ective techniques for detection and di?erentiation of embryogenic and non-embryogenic callus. A previous research using microarray technique had shown some potential candidate genes related to oil palm somatic embryogenesis, such as: IAA-amino acid hydrolase ILR1-like1 (ilr1), late embryogenesis abundant (lea2), 26S proteasome non-ATPase regulatory subunit 13 homolog B (26sp), and alpha trehalose phosphate synthase [UDP-forming] 6-like (tps6). The objective of this study was to analyze the transcription level of ilr1, lea2, 26sp, and tps6 using quantitative reverse transcription PCR (RT-qPCR). Non-embryogenic nodular callus and somatic embryo (coleoptile stage) of Elaeis guineensis var. Tenera (Deli Dura x AVROS Pisifera) leaf explants were collected from three palms for RNA extraction. The frst strand cDNA was synthesized from RNA and used for gene expression analysis. The expressions of four embryogenesis-related genes were analyzed using Relative Quantifcation Standart Curve method. RQ value was analyzed with One-way ANOVA and Dunnett’s test using Statistical Product and Service Solution (SPSS) 20.0 for windows. In RT-qPCR analysis, non-embryogenic nodular callus was used as calibrator sample and 40S ribosomal protein S27-2 (40s) was used as reference gene. The result shows that ilr1 and lea2 genes were signifcantly transcribed higher on coleoptile stage of the somatic embryo compared to callus, in other hand 26sp, and tps6 shows no expression di?erence on both samples.ilr1 genes gave the highest expression in somatic embryo compared to callus in most tested palms. Thus, it indicated that ilr1 may potentially involve in oil palm somatic embryogenesis and can be used as a candidate to develop the marker for embryogenesis in oil palm.

Proteomic identification of differentially expressed proteins during the acquisition of somatic embryogenesis in oil palm (Elaeis guineensis Jacq.)

In the present study we have identified and characterized the proteins expressed during different developmental stages of Elaeis guineensis calli obtained from zygotic embryos. We were interested in the possible proteomic changes that would occur during the acquisition of somatic embryogenesis and therefore samples were collected from zygotic embryos (E1), swollen explants 14days (E2) in induction medium, primary callus (E3), and pro-embryogenic callus (E4). The samples were grinded in liquid nitrogen, followed by total protein extraction using phenol and extraction buffer. Proteins were analyzed by two-dimensional electrophoresis (2-DE) and the differentially expressed protein spots were analyzed by MALDI-TOF mass spectrometry (MS and MS/MS). Interestingly, we have identified proteins, which can be used as potential candidates for future studies aiming at the development of biomarkers for embryogenesis acquisition and for the different stages leading to pro-embryogenic callus formation such as type IIIa membrane protein cp-wap13, fructokinase and PR proteins. The results obtained shed some light on the biochemical events involved in the process of somatic embryogenesis of E. guineensis obtained from zygotic embryos. The use of stage-specific protein markers can help monitor cell differentiation and contribute to improve the protocols for successfully cloning the species. Understanding the fate and dynamics of cells and tissues during callus formation is essential to understand totipotency and the mechanisms involved during acquisition of somatic embryogenesis (SE). In this study we have investigated the early stages of somatic embryogenesis induction in oil palm and have identified potential markers as well as proteins potentially involved in embryogenic competence acquisition. The use of these proteins can help improve tissue culture protocols in order to increase regeneration rates. This article is part of a Special Issue entitled: Environmental and structural proteomics.

The histodifferentiation events involved during the acquisition and development of somatic embryogenesis in oil palm (Elaeis guineensis Jacq.)

Understanding the fate and dynamics of cells during callus formation is essential to understanding totipotency and the somatic embryogenesis (SE) mechanisms. In the present study, the histodifferentiation events involved during the acquisition and development of somatic embryogenesis in oil palm (Elaeis guineensis Jacq.) was investigated. Zygotic embryos were inoculated on SE induction medium, and at 14 days the first divisions of the procambial and perivascular cells were observed. This region progressed to the formation of meristematic masses at 21 days, indicating their procambial and perivascular origin. Primary calli emerged at 45 days of culture, followed by progression to embryogenic calli at 90 days. The formation of proembryos (PE) from the meristematic cells occurred at 135 days of cultivation. The PE were isolated from the tissue of origin by the slight thickening of the cell wall, indicating their unicellular origin. When transferred to the maturation phase, differentiation of the somatic embryos at different developmental stages (globular and torpedo) was observed. The differentiated somatic embryos presented protoderm, procambial strands and plumules. Afterwards, they were transferred to culture medium without growth regulators in which conversion of the somatic embryos from torpedo stage into plants was observed. These results enable a greater understanding of the SE process and plantlet formation in E. guineensis.

New approaches to improve the efficiency of somatic embryogenesis in oil palm (Elaeis guineensis Jacq.) from mature zygotic embryos

We developed an efficient and simple system for inducing somatic embryogenesis and regenerating plantlets from mature zygotic embryos of oil palm. Embryogenic calli were induced from mature zygotic embryos of oil palm on modified Murashige and Skoog medium with 2,4-dichlorophenoxyacetic acid or picloram, alone or in combination with activated charcoal. The greatest frequency of embryogenic callus induction (97.5%) was obtained by culturing mature zygotic embryos on callus induction medium with 450 μM picloram and 2.5 g L−1 activated charcoal. Embryogenic calli proliferated on a medium with a reduced concentration of picloram. Embryogenic calli were then subcultured on a medium supplemented with 12.3 μM 2-isopentenyladenine and 0.54 μM naphthaleneacetic acid, with subcultures at 4-wk intervals. Somatic embryos were regenerated on a medium with Murashige and Skoog macro- and micronutrients at half-strength concentrations supplemented with 20 g L−1 sucrose, 2.5 g L−1 activated charcoal, and 2.5 g L−1 Phytagel. Detailed histological analysis revealed that somatic embryogenesis followed an indirect pathway. Primary calli were observed after 4–6 wk of culture and progressed to embryogenic calli at 12 wk. Embryogenic cells exhibited dense protoplasm, a high nucleoplasmic ratio, and small starch grains. Proembryos, which seemed to have a multicellular origin, formed after 16–20 wk of culture and successive cell divisions. Differentiated somatic embryos had a haustorium, a plumule, and the first and second foliar sheaths. In differentiated embryos, the radicular protrusion was not apparent because it generally does not appear until after the first true leaves emerge.

A candidate auxin-responsive expression marker gene, EgIAA9, for somatic embryogenesis in oil palm (Elaeis guineensis Jacq.)

This study correlates the potential for somatic embryogenesis in cultured leaf explants of oil palm trees with changes in the expression of hormone-responsive genes. The results show that expression of the putative Aux/IAA gene EgIAA9 is specifically correlated to somatic embryogenesis. A regression model for embryogenesis indicated a predictive R2 value of 63.55 % for this expression marker. Auxin is the plant growth regulator used for induction during the early stages of oil palm tissue culture. Expression of EgIAA9 decreased in response to increasing concentrations of exogenous 2,4-D. EgIAA9 expression profiles in cultured leaf explants from highly embryogenic palms, in embryogenic and non-embryogenic calli, and in response to increasing exogenous 2,4-D, suggested that endogenous auxin levels may be greater in highly embryogenic palms. The use of this marker to predict embryogenesis in oil palm tissue culture prior to the visible onset of this event is promising. Moreover, the results of this study prompt further investigations into the role of Aux/IAA genes in oil palm somatic embryogenesis.

In Vitro Callus Induction and Embryogenesis of Oil Palm (Elaeis guineensis Jacq.) from Leaf Explants

This research was to study in vitro callus induction and somatic embryogenesis in oil palm from leaf explants. Young leaf segments from mature oil palm were cultured on MS medium supplemented with different concentrations of 2,4-D with or without addition of 2 g/l activated charcoal (AC) or 2,4-D and picloram. Embryogenesis induction was done using MS medium containing 2,4-D 450 µM and benziladenine 4.4 µM with 3g/l activated charcoal. The treatment of 2,4-D 15 µM resulted in the highest percentage of callus induction. The treatment of 2,4-D and AC showed that 2,4-D 450 µM and AC led to higher percentage of callus induction than that of 2,4-D 400 µM and 2 g/l AC. Embryogenesis occured in 27 out of 250 clumps of primary callus was occurred after 2-3 times subcultures. Somatic embryo development occurred when the embryogenic callus was transferred on the same basal medium supplemented with casein hydrolysate with 1 µM BA or growth regulator free basal medium with 2 g/l activated charcoal.