Orchid is an important ornamental plant in Indonesia due to their natural beauty of flowers. In the tropical forest, orchids are being acquired for trading and commercial market. Thus, the effort is required to proliferate orchid in large quantities for conservation and improve the floral variation for plant breeding. The purpose of this study is to develop a firmed methodology of molecular breeding of orchids using CRISPR/Cas9 KO system. The plant material used was Phalaenopsis amabilis protocorms growth on NP medium+pepton (2 g/L). Protocorm were submerged in the culture of Agrobacterium tumefaciens that Ti‐plasmid had been filled with a T‐DNA construct of a pRGEB32 vector harboring sgRNA with PDS3 sequence. Detection for transformants was confirmed by PCR using HPT primers (545 bp), Cas9 primers (402 bp), PDS primers (280 bp) and trnL‐F (1200 bp) as an internal control. The results showed that 0.96% PDS transformants were obtained from PDS3T2 lines. Several transformant showed pale leaf color compared to non‐transformant plants. This study suggests that the target gene has successfully edited by CRISPR/Cas9 system and could be applied for that functional gene editing in orchids.
Dendrobium capra is an Indonesian endemic orchid species that live in Java. It grows on low altitude with warm climate. D. capra has beautiful small yellow greenish flower that grow in raceme inflorescence. This orchid faces a threat in its natural habitat due to having a long life cycle and a forestry main commodity as a main host thus categorized as Appendix II on CITES list. To address that problem, ex situ conservation approach using in vitro culture method is necessary. Germination enhancement effort using complex organic substances found that 200 ml/l tomato extract gave best germination result. Analysis on D. capra plantlet growth also showed that MS medium produced better plantlet size than NP, VW and KC medium. Supplementing medium with a combination of NAA and TDZ has also successfully induced early flowering within 11 month of culture period. This information is important to achieve successful in vitro culture of D. capra for various purposes.
Phalaenopsis amabilis (L.) Blume orchid is an Indonesian national flower. The number of these orchids in their natural habitat is very limited, therefore plant propagation efforts are needed. One of the promising methods is plant propagation by inserting embryo gene AtRKD4 from a model plant Arabidopsis thaliana into the orchid genome to produce many somatic embryos. From previous research, we have obtained 28 plant P. amabilis transformants carrying the AtRKD4 gene, however, it was unknown whether these plants have normal phenotypes and growth similar to their parents. Therefore, descriptions on growth and morphology are needed. This research aimed to evaluate the phenotype of P. amabilis carrying 35S::AtRKD4 the transformants grown in greenhouse. To achieve it, AtRKD4 gene integration stability on transformants genome was analyzed. Morphology and cross-section anatomy structure on transformant and non-transformant plantswere described. The stability of AtRKD4 gene integration in the plant genome was confirmed by amplification of the AtRKD4 gene from genomic DNA with Polymerase Chain Reaction (PCR) using a specific primer for AtRKD4 and ACTIN genes as the internal control. The quantitative data from morphology and anatomy measurements were analyzed statistically using ANOVA. The results showed that AtRKD4 was stably integrated into the genome of P. amabilis transformants and all transformant plants showed similar morphology and anatomy characteristics as non-transformant plants. The AtRKD4 embryo gene was stably integrated into the orchid genome and the transformant plants grow normally without significant changes in phenotype.
Here we describe a novel method of using green fluorescence protein (GFP) as a reporter gene for early detection of an integrated TDNA containing the orchid flowering gene, PaFT1 (Phalaenopsis aphrodite Flowering locus T1) in the tobacco genome. Functional assays that report the presence of exogenous DNA early in development are especially useful in plants where the desired phenotype is only apparent after long periods of vegetative growth. The objective of this study is to establish a method for detecting an inserted Phalaenopsis orchid flowering gene and examining its function in tobacco. The p35S::PaFT1 35S::GFP construct was introduced into Agrobacterium tumefaciens strain EHA101. Transformed tobacco leaves were cultured on MS medium with addition of 1 mgL 1 NAA+3 mgL 1 BAP+50 mgL 1 Kanamycin+300 mgL 1 timentin for selection. Results showed bright green GFP fluorescent signals in 11 out of 15 (73%) tobacco leaf cells at a 2month time point after transformation. GFP and PaFT1 fragments were amplified in genomic PCR using GFP and PaFT1 specific primers. The accumulated PaFT1 transcripts were observed in 3 monthold transgenic tobacco plants containing p35S::PaFT135S::GFP. Green florescence was observed only in the transgenic plants at the 5 monthold stage but not in the wild type controls.
Malaria is a major infectious disease in the world. The disease is caused by blood protozoan from the genus Plasmodium. The main problem in controling this disease is resistance parasite cases to drugs that have been used. Cucumber (Cucumis sativa L.) contain bioactive compounds suspected of terpenoids and saponins are believed to reduce the level of parasitemia. Waste of skin and cucumber base is very abundant, especially from stalls in the city of Yogyakarta. Estimated at the base and cucumber skin there are bioactive content that can be used as an alternative antimalarial drug. This research aims to study the potential of chloroform extract and the base of the fruit peel waste to the level of parasitemia of Plasmodium berghei in mice.First step is collecting the waste of skin and cucumber base in some stalls in the city of Yogyakarta. Extraction by maceration method using chloroform solvent, the method further phytochemical studies by Thin Layer Chromatography (TLC). Antiplasmodium test with negative control treatment (DMSO 0.3%), positive control (chloroquine 3 mg / kg), the dose C.sativa extract 100; 200; 300; 400 and 500 mg / kg in male mice given strain Switzerland 3 months of age infected with Plasmodium berghei orally. T he results showed there were terpenoids and saponins compounds in the chloroform extracts of C.sativa base and skin. The most effective dose of the extract inhibition of parasitemia level P. berghei in mice treated P5 is the highest (500 mg / kg BB), which is still higher than the standard drug Chloroquine so that waste of skin and the base C.sativa potential as an alternative antimalarial drug.Key words: Waste of skin and the base of cucumber (Cucumis sativa L.) Antimalarial.
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