Understanding the molecular mechanisms underlying somatic embryogenesis is essential for resolving the problems related to the long duration of the process and a low rate of somatic embryo induction in oil palm tissue culture. In this study, we conducted genome-wide identification of the oil palm homeodomain leucine zipper (EgHD-ZIP) family, which is one of the plant-specific transcription factors reported to be involved in embryogenesis. EgHD-ZIP proteins can be divided into four subfamilies, which have similarities in gene structure and protein-conserved motifs within a group. In silico expression analysis showed that the expression of EgHD-ZIP gene members in the EgHD-ZIP I and II families, as well as most members in the EgHD-ZIP IV family, were up-regulated during the zygotic and somatic embryo developmental stages. In contrast, the expression of EgHD-ZIP gene members in the EgHD-ZIP III family was down-regulated during zygotic embryo development. Moreover, the expression of EgHD-ZIP IV genes was validated in the oil palm callus and at the somatic embryo stages (globular, torpedo, and cotyledon). The results revealed that EgHD-ZIP IV genes were up-regulated at the late stages of somatic embryogenesis (torpedo and cotyledon). While BABY BOOM (BBM) gene was up-regulated at the early stage of somatic embryogenesis (globular). In addition, the Yeast-two hybrid assay revealed the direct binding between all members of the oil palm HD-ZIP IV subfamily (EgROC2, EgROC3, EgROC5, EgROC8, and EgBBM). Our findings suggested that the EgHD-ZIP IV subfamily and EgBBM work together to regulate somatic embryogenesis in oil palms. This process is important because it is widely used in plant biotechnology to produce large quantities of genetically identical plants, which can be used for oil palm tissue culture improvement.

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.

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Transient gene expression is an important technique in gene functional analysis, protein production and in plants. However, traditional transient expression methods using Agrobacterium are time-consuming with low efficiency. In this study, we demonstrated the use of a single-walled carbon nanotube (SWCNT) to deliver 35S:mCherry:pCXSN plasmid into rice calli. This transient expression protocol used a plastic medical syringe to create the physical pressure to help the delivery of plasmid DNA into plant cells. This protocol is relatively easy to perform and low cost. The transient expression was observed under fluorescence microscopy, and the mCherry fluorescence signal was quantified. The plasmid DNA was delivered into the rice cell using a 3:1 ratio (plasmid: carbon nanotube). The result showed that the mCherry signal of carbon nanotube + plasmid DNA treatment was the highest signal at 3 days post-transformation and decreased to a lower signal at 6 days post-transformation. Moreover, the quantitative analysis of mCherry mean intensity revealed that the signal intensity of carbon nanotube + plasmid DNA treatment was the highest level, and significantly higher than the control treatments at 3 days post-transformation. Plasmid DNA can be transported easily into plant calli using this carbon nanotube transient  expression protocol.