Kultur suspensi sel tanaman gajah beranak (Goniothalamus tapis Miq) terhadap kandungan zat goniotalamin

Zat goniotalamin pada tanaman gajah beranak (Goniothalamus tapis) merupakan obat alternatif penyembuhan kanker. Penelitian bertujuan untuk mendapatkan zat goniotalamin melalui kultur kalus dan kultur suspensi sel. Metode penelitian eksperimen Rancangan Acak Lengkap (RAL) dengan kombinasi 2,4-D (1-10 mgL-1) dan BAP (0,5-2 mgL-1) menggunakan eksplan batang muda, terdiri dari 17 perlakuan dan 3 kali ulangan. Analisis data menggunakan Analysis of Variances dan uji lanjut Duncan Multiple Range Test (DMRT) taraf 5%. Hasil menunjukkan bahwa kultur kalus G. tapis pada media 5,0 mg L-1 2,4-D + 1 mg L-1 BAP adalah yang terbaik dengan waktu muncul kalus 28,33 hari dan persentase pembentukan kalus 100%. Kalus untuk kultur suspensi sel bertekstur remah dan berwarna kuning kehijauan. Kultur suspensi sel menghasilkan pertumbuhan sel yang cepat, tidak lembek berair dan mudah dipisahkan. Hasil kualitatif Kromatografi Lapis Tipis kultur suspensi sel sangat jelas, bersih dan terdapat potensi kandungan zat goniotalamin pada perlakuan 2,4-D 5 mg L-1 + BAP 0,5 mg L-1, 2,4-D 5 mg L-1 + BAP 1 mg L-1, 2,4-D 5 mg L-1 + BAP 2 mg L-1, 2,4-D 10 mg L-1 + BAP 0,5 mg L-1 dan 2,4-D 10 mg L-1 + BAP 1 mg L-1. Hasil kuantitatif zat goniotalamin dengan Kromatografi Cair Prestasi Tinggi terdapat pada perlakuan 2,4-D 5,0 mgL-1 + BAP 1 mg L-1 yaitu 9,57 mg g-1.The goniothalamine compound on Goniothalamus tapis is an alternative cancer medicine. This study aimed to obtain gonotalamin through callus culture and suspension cell culture. The experiment research method was Completely Randomized Design (CRD) with a combination of 2.4-D (1-10 mg L-1) and BAP (0.5-2 mg L-1) using young stem explants consisting of 17 treatments with 3 replications. Data analysis used ANOVA and DMRT at 5%. The results showed that G. tapis callus culture on 5.0 mg L-1 2.4-D + 1 mg L-1 BAP was the best treatment medium with callus emergence time of 28.33 days and percentage of callus formation 100%. The callus used for suspension cell culture was friable and greenish-yellow in color. Suspension cell culture resulted in rapid cell growth, was not fleshy, and easily separated. The quality test by Thin Layer Chromatography (TLC) from suspension cell culture resulted very clear, clean, and potential content of goniothalamin found in treatments 2.4-D 5.0 mg L-1 + BAP 0.5 mg L-1, 2.4-D 5.0 mg L-1 + BAP 1 mg L-1, 2.4-D 5.0 mg L-1 + BAP 2 mg L-1, 2.4-D 10 mg L-1 + BAP 0.5 mg-1 and 2.4-D 10 mg-1 + BAP 1 mg-1. The quantitative results of the best goniotalamine compounds in cell suspension cultures using High-Performance Liquid Chromatography (HPLC) on medium 2,4-D 5.0 mgL-1 + BAP 1 mg L-1 ie 9.57 g-1.

Assessment of the Level of Accumulation of the dIFN Protein Integrated by the Knock-In Method into the Region of the Histone H3.3 Gene of Arabidopsis thaliana

Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We investigated the possibility of obtaining a suspension cell culture of Arabidopsis thaliana carrying a site-specific integration of a target gene encoding modified human interferon (dIFN) using endonuclease Cas9. For the targeted insertion, we selected the region of the histone H3.3 gene (HTR5) with a high constitutive level of expression. Our results indicated that Cas9-induced DNA integration occurred with the highest frequency with the construction with donor DNA surrounded by homology arms and Cas9 endonuclease recognition sites. Among the monoclones of the four cell lines with knock-in studied, there is high heterogeneity in the level of expression and accumulation of the target protein. The accumulation of dIFN protein in cell lines with targeted insertions into the target region of the HTR5 gene does not statistically differ from the level of accumulation of dIFN protein in the group of lines with random integration of the transgene. However, one among the monoclonal lines with knock-in has a dIFN accumulation level above 2% of TSP, which is very high.

Pharmacokinetic Study of Anti-osteoarthritic Compounds of a Standardized Fraction from Sphaeralcea Angustifolia

Sphaeralcea angustifolia has been widely used in inflammatory conditions such as blows, bruises, fractures, and wounds. The compounds identified as active in plants and suspension cell culture of S. angustifolia were tomentin, scopoletin, and sphaeralcic acid. To consolidate the integral use of knowledge about the S. angunstifolia and strengthen its pharmacological use in patients with knee osteoarthritis, the pharmacokinetic behavior of the active compounds was characterized. The SaTSS (S. angustifoloia standardized in Tomentin, Scopoletin, and Sphaeralcic acid) anti-ostearthritic fraction was obtained from cell suspension. The analytical method of High-Performance Liquid Chromatography (HPLC) for tomentin, scopoletin, and sphaeralcic acid were validated determining the accuracy, precision linearity, sensibility, specificity, detection limits, and quantification time-range parameters, as well as extraction efficiency and stability of compounds. The pharmacokinetic assay was performed with ICR mice strain, in which the mice were administrated with a single oral or intravenous dose (400 mg/kg with 7.1 mg/kg of scopoletin and tomentin in mixture and 34.6 mg/kg of sphaeralcic acid) of the SaTSS standardized active fraction. The results of the validated analytical methods allowed establishing, in a validated manner, that a coumarin mixture and sphaeralcic acid present in the SaTES fraction were detected in plasma. According to the values of Akaike Information Criteria (AIC), Sum of Squares (SS), Schwarz Criteria (SC), and by the determination coefficient (R2), the compounds follow a two-compartment model.

Citrus Cell Suspension Culture Establishment, Maintenance, Efficient Transformation and Regeneration to Complete Transgenic Plant

Agrobacterium-mediated transformation of epicotyl segment has been used in Citrus transgenic studies. The approach suffers, however, from limitations such as occasionally seed unavailability, the low transformation efficiency of juvenile tissues and the high frequency of chimeric plants. Therefore, a suspension cell culture system was established and used to generate transgenic plants in this study to overcome the shortcomings. The embryonic calli were successfully developed from undeveloped ovules of the three cultivars used in this study, “Sweet orange”-Egyptian cultivar (Citrus sinensis), “Shatangju” (Citrus reticulata) and “W. Murcott” (Citrus reticulata), on three different solid media. Effects of media, genotypes and ages of ovules on the induction of embryonic calli were also investigated. The result showed that the ovules’ age interferes with the callus production more significantly than media and genotypes. The 8 to 10 week-old ovules were found to be the best materials. A cell suspension culture system was established in an H+H liquid medium. Transgenic plants were obtained from Agrobacterium-mediated transformation of cell suspension as long as eight weeks subculture intervals. A high transformation rate (~35%) was achieved by using our systems, confirming BASTA selection and later on by PCR confirmation. The results demonstrated that transformation of cell suspension should be more useful for the generation of non-chimeric transgenic Citrus plants. It was also shown that our cell suspension culture procedure was efficient in maintaining the vigor and regeneration potential of the cells.

Expression of a Bacterial Trehalose-6-phosphate Synthase otsA Increases Oil Accumulation in Plant Seeds and Vegetative Tissues

We previously demonstrated that exogenous trehalose 6-phosphate (T6P) treatment stabilized WRINKLED1 (WRI1), a master transcriptional regulator of fatty acid (FA) synthesis and increased total FA content in Brassica napus (B. napus) embryo suspension cell culture. Here, we explore Arabidopsis lines heterologously expressing the Escherichia coli T6P synthase (otsA) or T6P phosphatase (otsB) to refine our understanding regarding the role of T6P in regulating fatty acid synthesis both in seeds and vegetative tissues. Arabidopsis 35S:otsA transgenic seeds showed an increase of 13% in fatty acid content compared to those of wild type (WT), while seeds of 35:otsB transgenic seeds showed a reduction of 12% in fatty acid content compared to WT. Expression of otsB significantly reduced the level of WRI1 and expression of its target genes in developing seeds. Like Arabidopsis seeds constitutively expressing otsA, transient expression of otsA in Nicotiana benthamiana leaves resulted in strongly elevated levels of T6P. This was accompanied by an increase of 29% in de novo fatty acid synthesis rate, a 2.3-fold increase in triacylglycerol (TAG) and a 20% increase in total fatty acid content relative to empty vector (EV) controls. Taken together, these data support the heterologous expression of otsA as an approach to increasing TAG accumulation in plant seeds and vegetative tissues.

Suspension Cell Culture of Dioscorea deltoidea—A Renewable Source of Biomass and Furostanol Glycosides for Food and Pharmaceutical Industry

Dioscorea deltoidea is a medicinal plant valued for its high content of steroidal glycosides (SG)—bioactive compounds with cardioprotective and immunomodulation actions, also used to treat reproductive system disorders. To overcome the limitations of natural resources of this species, a suspension cell culture of D. deltoidea was developed as a renewable and ecologically sustainable source of raw biomass and SG. Cell culture demonstrated stable and intensive growth in the laboratory (20 L) and industrial (630 L) bioreactors operated under a semi-continuous regime (specific growth rate 0.11–1.12 day−1, growth index 3.5–3.7). Maximum dry weight accumulation (8.5–8.8 g/L) and SG content (47–57 mg/g DW) were recorded during the stationary phase. Bioreactor-produced cell biomass contained inorganic macro (K, Ca, Mg, Na) and micro (Zn, Mn, Fe, B, Al, Cu, Cr, Se, Co, Ni) elements in concentrations within the safe range of dietary recommendations. Acute toxicity test showed no or insignificant changes in organ weight, hematological panel and blood biochemistry of laboratory animals fed with 2000 and 5000 mg/kg dry biomass. The results suggest that cell culture of D. deltoidea grown in bioreactors has great potential to be used as functional foods and a component of specialized dietary supplements in complex therapy of reproductive system disorders and mineral deficiency.