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.

Totipotency of Daucus carota L. Somatic Cells Microencapsulated Using Spray Drying Technology

The carrot is considered a model system in plant cell culture. Spray drying represents a widely used technology to preserve microorganisms, such as bacteria and yeasts. In germplasm conservation, the most used methods are freeze drying and cryopreservation. Therefore, the aim of this work was to evaluate the effect of spray drying on the viability and totipotency of somatic carrot cells. Leaf, root and stem explants were evaluated to induce callus with 2 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D). Calli obtained from the stem were cultivated in a liquid medium with 1 mg/L of 2,4-D. Cell suspensions were spray dried with maltodextrin-gum Arabic and maltodextrin-xanthan gum mixtures, two outlet air temperatures (50 and 60 °C) and 120 °C inlet air temperature. Results showed that carrot cells were viable after spray drying, and this viability remained for six months at 8 °C. The totipotency of the microencapsulated cells was proven. Cells that were not spray dried regenerated 24.6 plantlets, while the spray dried cells regenerated 19 plantlets for each gram of rehydrated powder. Thus, spray drying allowed researchers to obtain viable and totipotent cells. This work is the first manuscript that reported the spray drying of plant somatic cells.

In vitro selection of birch for tolerance to salinity stress

In vitro modelling of stress is one of the promising avenues for plant breeding for tolerance to negative environmental factors. In this study we examined the effect of NaCl (0.5%) on callusogenesis and morphogenesis of stem explants of different birch genotypes: Betula pendula Roth, B. pendula Roth var. carelica (Mercklin) Hämet-Ahti, B. pendula f. ‘dalecarlica’ (L.f.) Schneid., B. pubescens Ehrh. In our experiments we used pre-selected microclones from our in vitro collection on NaCl (0.2-1.0%) selective media. The clones were contrasted by the degree of their sensitivity to salinity (so-called ‘stable’ and ‘sensitive’ microclones). With the use of stem callus cultures we identified informative, simple and reproducible indicators for the selection of salt-tolerant genotypes. Among these indicators were the frequency of callus formation and the viability of callus cultures, which were significantly higher in ‘stable’ group of microclones. Polyploid birch clones (2n=4x=56, 2n=3x=42) were more resistant to salination compared to diploid clones (2n=28). Our study has shown that the selection of salt-tolerant birch lines can be based on the plants’ genetic diversity presented in the collection (various species, varieties, hybrids, polyploids) and manifested in the process of in vitro cultivation, as well as in the cellular heterogeneity of callus cultures.

Protoplast Isolation, Fusion, Culture and Transformation in the Woody Plant Jasminum spp.

Plant protoplasts are significant for plant cell culture, somatic cell fusion, genetics, and breeding studies. In addition, in vitro plant regeneration has great importance for developmental biology, manifesting potential applications in agriculture and biotechnology. In this regard, we present a well-established protocol regarding protoplast isolation, cell culture and protoplast fusion of Jasminum spp. In particular, different tissues of Jasminum samab L. and Jasminum mesnyi were employed for protoplast isolation, and stem explants provided a high callus induction rate in a short period of time. The best source for protoplast isolation was calli tissues. The optimized isolation protocol consisted of digesting callus in an enzyme solution containing 0.4 M mannitol, 0.2 M MES, 1 M CaCl2, 0.2 M KCL and 1 M NaH2PO4, 1.5% Cellulases onozuka R-10, 0.4% Macerozyme R-10 and 0.8% Pectinase for 4 h at 26 °C in the dark, providing a yield of 23.8 × 106 Protoplast/gFW with 88% viability. Protoplasts were cultured both in liquid and agarose medium under optimum conditions, leading to microcalli formation after eight weeks. A 5% protoplast-fusion rate can be achieved when cultured in 40% (w/v) PEG-MW6000 supplemented with 0.1 M CaCl2, 0.1 M sorbitol and 1 M Tris for 20 min. Furthermore, we developed an efficient PEG-mediated transformation protocol for jasmine protoplasts. The best results regarding protoplast transformation were obtained when the protoplast concentration was 4 × 105 cells/mL and the exogenous plasmid DNA added had a concentration of 10 µg DNA/100 µL protoplast solution, followed by the application of 40% PEG-4000 for 10 min.

Callus Induction of Leaves and Stems in Krisan (Chrysanthemum morifolium Ramat cv Dewi ratih) with Alternative Foliar Fertilizers Media

Availability of quality seeds in production of krisan (Chrysanthemum morifolium Ramat cv Dewi ratih) cultivation is still rare, therefore research on seed multiplication through tissue culture is needed. The media used in tissue culture is relatively expensive for home industry. This study aims to determine the respond of leaf and stem explants using foliar fertilizers (Growmore, Gandasil D and Mutiara) as an alternative media for callus inductions. This study used a Completely Randomized Design (CRD) consisted of 4 treatments: P0: ½ MS + 0,25 mg/l BAP, P1 (Growmore + 0,25 mg/l BAP), P2 (Gandasil D + 0,25 mg/l BAP), P3 (Mutiara + 0,25 mg/l BAP). The variables observed in this study included callus appearance time, callus color and callus texture. The result of this study indicated that the use of BAP (6-Benzyl Amino Purine) affected the time of callus formation and callus morphology. Callus was formed on leaf explants 13 days after planting while on stem explants 7 days after planting and compact texture. Growmore + 0,25 mg/l BAP treatment yields the best callus on leaf explant, while Gandasil D + 0,25 mg/l BAP treatment yields the best callus on stem explant.

Transcriptome Dynamics of Epidermal Reprogramming during Direct Shoot Regeneration in Torenia fournieri

Shoot regeneration involves reprogramming of somatic cells and de novo organization of shoot apical meristems (SAMs). In the best-studied model system of shoot regeneration using Arabidopsis, regeneration occurs mediated by auxin-responsive pluripotent callus formation from pericycle or pericycle-like tissues according to the lateral root development pathway. In contrast, shoot regeneration can be induced directly from fully differentiated epidermal cells of stem explants of Torenia fournieri (Torenia) without intervening callus mass formation in culture with cytokinin, yet its molecular mechanisms remain unaddressed. Here we characterized this direct shoot regeneration by cytological observation and transcriptome analyses. The results showed that the gene expression profile rapidly changes upon culture to acquire a mixed signature of multiple organs/tissues, possibly associated with epidermal reprogramming. Comparison of transcriptomes between three different callus-inducing cultures (callus induction by auxin, callus induction by wounding, and protoplast culture) of Arabidopsis and the Torenia stem culture identified genes upregulated in all the four culture systems as candidates of common factors of cell reprogramming. These initial changes proceeded independently of cytokinin, followed by cytokinin-dependent, transcriptional activations of nucleolar development and cell cycle. Later, SAM regulatory genes became highly expressed, leading to SAM organization in the foci of proliferating cells in the epidermal layer. Our findings revealed three distinct phases with different transcriptomic and regulatory features during direct shoot regeneration from the epidermis in Torenia, which provides a basis for further investigation of shoot regeneration in this unique culture system.

Iron-doped zinc oxide nanoparticles-triggered elicitation of important phenolic compounds in cell cultures of Fagonia indica.

Fagonia indica is an important medicinal plant species used traditionally against a variety of diseases. In this study, we initiated callus cultures from healthy stem explants. We observed maximum callus induction frequency (88%) on MS media supplemented with Thidiazuron (1.0 mg/mL). We also examined the callus cultures to determine the impact of iron-doped zinc oxide nanoparticles (Fe-ZnO-NPs) in concentrations (15.62 to 250 µg/mL) on biomass accumulation, secondary metabolism, and antioxidative potential in callus cultures of F. indica. Our results showed that maximum callus biomass (FW = 13.6 g and DW = 0.58 ± 0.01) was produced on day 40 when the media was supplemented with 250 µg/mL Fe-ZnO-NPs. Similarly, maximum TPC (268.36 µg GAE/g of DW) was detected in 40 days old callus added with 125 µg/mL Fe-ZnO-NPs. Maximum TFC (78.56 µg QE/g of DW) was observed in 20 days old callus grown in 62.5 µg/mL Fe-ZnO-NPs containing media. Maximum total antioxidant capacity (390.74 µg AAE/g of DW) was observed in 40 days old callus with 125 µg/mL Fe-ZnO-NPs treated cultures, respectively. The antioxidant potential was in correlation with the TPC. These results showed that Fe-ZnO-NPs elicitors can increase the biomass and activate secondary metabolism in F. indica cells.

A novel micropropagation of Lycium ruthenicum and epigenetic fidelity assessment of three types of micropropagated plants in vitro and ex vitro

Lycium ruthenicum is an excellent eco-economic shrub. Numerous researches have been conducted for the function of its fruits but scarcely focused on the somaclonal variation and DNA methylation. An efficient micropropagation protocol from leaves and stems of L. ruthenicum was developed in this study, in which not only the leaf explants but also the stem explants of L. ruthenicum were dedifferentiated and produced adventitious buds/multiple shoots on one type of medium. Notably, the efficient indirect organogenesis of stem explants was independent of exogenous auxin, which is contrary to the common conclusion that induction and proliferation of calli is dependent on exogenous auxin. We proposed that sucrose supply might be the crucial regulator of stem callus induction and proliferation of L. ruthenicum. Furthermore, results of methylation-sensitive amplified polymorphism (MSAP) showed that DNA methylation somaclonal variation (MSV) of CNG decreased but that of CG increased after acclimatization. Three types of micropropagated plants (from leaf calli, stem calli and axillary buds) were epigenetically diverged more from each other after acclimatization and the ex vitro micropropagated plants should be selected to determine the fidelity. In summary, plants micropropagated from axillary buds and leaves of L. ruthenicum was more fidelity and might be suitable for preservation and propagation of elite germplasm. Also, leaf explants should be used in transformation. Meanwhile, plants from stem calli showed the highest MSV and might be used in somaclonal variation breeding. Moreover, one MSV hotspot was found based on biological replicates. The study not only provided foundations for molecular breeding, somaclonal variation breeding, preservation and propagation of elite germplasm, but also offered clues for further revealing novel mechanisms of both stem-explant dedifferentiation and MSV of L. ruthenicum.