Peranan Vitamin C dan Acetosyringone pada Transformasi Genetik Anggrek Vanda Tricolor Lindl. Var. Suavis melalui Agrobacterium Tumefaciens

Vanda tricolor Lindl. var. suavis adalah spesies anggrek alam asli Indonesia yang di habitat asalnyasudah mulai langka karena kerusakan hutan. Pengembangan metode transfer gen untuk tujuan modifikasigenetik diperlukan baik untuk perbaikan sifat-sifat tanaman maupun konservasi. Metode transfer genmelalui Agrobacterium tumefaciens untuk anggrek V. tricolor belum ada protokolnya, sehingga perludikembangkan. Penelitian ini menggunakan protokorm Vanda tricolor Lindl. forma Bali yang berumur8 minggu setelah semai sebagai target transformasi. T-DNA membawa gen KNAT1 yang dikontrololeh promoter CaMV dari Cauliflower Mosaic Virus serta membawa gen NPTII, yakni gen ketahananterhadap antibiotik kanamisin untuk seleksi transforman. T-DNA ini dikonstruksi dalam vektor binerPgreen dan ditransfer ke genom tanaman melalui A. tumefaciens stranin LBA4404. Deteksi transgendilakukan dengan PCR untuk mengamplifikasi fragmen gen KNAT1 sepanjang 1200bp dengan primerspesifik KNAT1. Tujuan dari penelitian ini adalah: 1) mengetahui peran vitamin C pada transformasianggrek V. tricolor dalam mengatasi browning (pencoklatan); 2) menentukan pada tahapan mana(inokulasi dan atau kokultivasi) acetosyringone perlu ditambahkan dalam proses transformasi; dan 3)menentukan perlakuan yang menghasilkan persentase kandidat transforman dan frekuensi transformasitertinggi. Hasil penelitian mendapatkan bahwa aplikasi vitamin C mencegah terjadinya browning padaprotokorm anggrek V. tricolor yang ditransformasi meningkatkan persentase protokorm hijau setelahseleksi transforman. Persentase kandidat transforman dan frekuensi transformasi tertinggi diperoleh jikaacetosyringone diaplikasikan pada tahap inokulasi dan kokultivasi, kemudian diberi perlakuan vitaminC setelah kokultivasi

Genotypic And Phenotypic Characterization Of Alcaligenes Javaensis Jg3 Potential AS An Effective Biodegrader

Utilization of glycerol by lipase producing bacteria offers great benefits for fat and oil waste degradation and waterwaste treatment. Nevertheless, there have been lack of reports about the availability of non-pathogenic, lipase producing bacteria, which could naturally degrade glycerol produced from the lipolysis process by lipase. This study reported a newly identified species of rhizobacteria, Alcaligenes javaensis JG3, which is not only able to produce high level of lipase, but also able to degrade glycerol molecules. Identification of strain JG3 was carried out using SEM (Scanning Electron Microscope), BD Phoenix 100 Automated Microbiology System and 16S rRNA gene analysis to determine its taxonomy status. The ability of the strain to metabolize glycerol was investigated both genotypically and phenotypically using degenerate PCR and a glycerol minimal medium. Identification test results showed that strain JG3 belongs to genus Alcaligenes, with the closest relationship with A. faecalis and A. aquatilis (96% nucleotide similarity maximum). Degenerate PCR resulted in a 248-bp sequence showing 93% similarity with glpK of Candidatus Sodalis pierantonius SOPE, a key gene involved in glycerol metabolism. In vitro glycerol utilization test result showed that Alcaligenes sp. JG3 was able to grow on glycerol aerobically, but not anaerobically. It is concluded that Alcaligenes sp. JG3 possesses genes coding for glycerol metabolism and this trait is phenotypically expressed, thus making the strain potential to be used as an effective fat and oil biodegrader

Dynamic Regulation of H3K27 Trimethylation during Arabidopsis Differentiation

During growth of multicellular organisms, identities of stem cells and differentiated cells need to be maintained. Cell fate is epigenetically controlled by the conserved Polycomb-group (Pc-G) proteins that repress their target genes by catalyzing histone H3 lysine 27 trimethylation (H3K27me3). Although H3K27me3 is associated with mitotically stable gene repression, a large fraction of H3K27me3 target genes are tissue-specifically activated during differentiation processes. However, in plants it is currently unclear whether H3K27me3 is already present in undifferentiated cells and dynamically regulated to permit tissue-specific gene repression or activation. We used whole-genome tiling arrays to identify the H3K27me3 target genes in undifferentiated cells of the shoot apical meristem and in differentiated leaf cells. Hundreds of genes gain or lose H3K27me3 upon differentiation, demonstrating dynamic regulation of an epigenetic modification in plants. H3K27me3 is correlated with gene repression, and its release preferentially results in tissue-specific gene activation, both during differentiation and in Pc-G mutants. We further reveal meristem- and leaf-specific targeting of individual gene families including known but also likely novel regulators of differentiation and stem cell regulation. Interestingly, H3K27me3 directly represses only specific transcription factor families, but indirectly activates others through H3K27me3-mediated silencing of microRNA genes. Furthermore, H3K27me3 targeting of genes involved in biosynthesis, transport, perception, and signal transduction of the phytohormone auxin demonstrates control of an entire signaling pathway. Based on these and previous analyses, we propose that H3K27me3 is one of the major determinants of tissue-specific expression patterns in plants, which restricts expression of its direct targets and promotes gene expression indirectly by repressing miRNA genes

In planta transformation method for T-DNA transfer in orchids

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Identification of early flowering mutant gene in Phalaenopsis amabilis (L.) Blume for sgRNA construction in CRISPR/Cas9 genome editing system

Phalaenopsis amabilis (L.) Blume commonly called Moth Orchid (Orchidaceae) is a natural orchid species designated as the National Flower of Indonesia for its beautiful flower shape and long-lasting flowering period. Basically, P. amabilis has a long vegetative phase that cause late flowering, about 2 to 3 years for flowering, hence a method to shorten vegetative period is desired. The latest technological approach that can be used to accelerate flowering of P. amabilis is the CRISPR/Cas9 genome editing method to inactivate the GAI (Gibberellic Acid Insensitive) gene as a mutant gene that can accelerate the regulation of FLOWERING TIME (FT) genes flowering biosynthesis pathway. The approach that needs to be taken is to silence the GAI gene with a knockout system which begins with identifying and characterizing the GAI target gene in the P. amabilis which will be used as a single guide RNA. CRISPR/Cas9 mediated knockout efficiency is highly dependent on the properties of the sgRNA used. SgRNA consists of a target sequence, determining its specificity performance. We executed phylogenetic clustering for the PaGAI protein with closely related orchid species such as Dendrobium capra, Dendrobium cultivars and Cymbidium sinensis. SWISS-Model as tool webserver for protein structure homology modeling. Results show that P. amabilis has a specific domain with the occurrence of point mutations in the two conservative domains. Therefore, a single guide RNA reconstruction needs to be implemented. © 2024, Instituto Internacional de Ecologia. All rights reserved