Cell Suspension Cultures Research Articles

Establishment of an efficient cell suspension culture system for Lonicera japonica Thunb

Establishment of an efficient cell suspension culture system for Lonicera japonica Thunb

Biotechnology for propagation and secondary metabolite production in Bacopa monnieri.

Bacopa monnieri (L.) Wettst. or water hyssop commonly known as "Brahmi" is a small, creeping, succulent herb from the Plantaginaceae family. It is popularly employed in Ayurvedic medicine as a nerve tonic to improve memory and cognition. Of late, this plant has been reported extensively for its pharmacologically active phyto-constituents. The main phytochemicals are brahmine, alkaloids, herpestine, and saponins. The saponins include bacoside A, bacoside B, and betulic acid. Investigation into the pharmacological effect of this plant has thrived lately, encouraging its neuroprotective and memory supporting capacity among others. Besides, it possesses many other therapeutic activities like antimicrobial, antioxidant, anti-inflammatory, gastroprotective properties, etc. Because of its multipurpose therapeutic potential, it is overexploited owing to the prioritization of natural remedies over conventional ones, which compels us to conserve them. B. monnieri is confronting the danger of extinction from its natural habitat as it is a major cultivated medico-botanical and seed propagation is restricted due to less seed availability and viability. The ever-increasing demand for the plant can be dealt with mass propagation through plant tissue culture strategy. Micropropagation utilizing axillary meristems as well as de novo organogenesis have been widely investigated in this plant which has also been explored for its conservation and production of different types of secondary metabolites. Diverse in vitro methods such as organogenesis, cell suspension, and callus cultures have been accounted for with the aim of production and/or enhancement of bacosides. Direct shoot-organogenesis was initiated in excised leaf and internodal explants without any exogenous plant growth regulator(s) (PGRs), and the induction rate was improved when exogenous cytokinins and other supplements were used. Moreover, biotechnological toolkits like Agrobacterium-mediated transformation and the use of mutagens have been reported. Besides, the molecular marker-based studies demonstrated the clonal fidelity among the natural and in vitro generated plantlets also elucidating the inherent diversity among the natural populations. Agrobacterium-mediated transformation system was mostly employed to optimize bacoside biosynthesis and heterologous expression of other genes. The present review aims at depicting the recent research outcomes of in vitro studies performed on B. monnieri which include root and shoot organogenesis, callus induction, somatic embryogenesis, production of secondary metabolites by in vitro propagation, acclimatization of the in vitro raised plantlets, genetic transformation, and molecular marker-based studies of clonal fidelity. KEY POINTS: • Critical and up to date records on in vitro propagation of Bacopa monnieri • In vitro propagation and elicitation of secondary metabolites from B. monnieri • Molecular markers and transgenic studies in B. monnieri.

Biomass Accumulation of Gynostemma Pentaphyllum (Thunb.) Makino in Cell Suspension Cultures inhibiting Human Cancer Cell Growth

Gynostemma pentaphyllum (Thunb.) Makino (GpM) is a medicinal plant in traditional medicine throughout Asia for the treatment of several diseases including cancer. GpM plant cell suspension cultures provide a time and cost effective well-controlled means promising a high-yielding biomass production of pharmaceutical compounds. The purpose of the current work is to investigate the effect of GpM cell suspension cultures on human cancer cell lines growth. The biomass was produced by cell suspension culture of GpM callus into 250 mL Erlenmeyer flask containing 50 mL of liquid medium culture. Gypenosides in GpM were confirmed by HPLC. Pharmacological activities of GpM extract were tested on human cancer cell line (HepG2, Huh7, A549 and HL-60) using Sulforhodamine B (SRB) cytotoxicity assay and Tetrazolium (MTT) assay. We successfully produced 5.485 ± 0.223 g GpM fresh biomass per 250 mL Erlenmeyer flask after 18 days culture. Total gypenosides and Rb1 in dry cell suspension were 48.844 ± 3.933 mg/g and 0.041 ± 0.004 mg/g. The crude extract from GpM cell suspension cultures exhibited significant cancerous cell growth inhibition in a dose dependent manner. From the MTT assay and SRB cytotoxicity assay, it is obvious that GpM cell suspension culture extract at 200 μg/mL significantly inhibited the growth of multiple human cancer cells including hepatoma cell lines (HepG2, Huh7), lung carcinoma cell line (A549) and leukemia cell line (HL-60). Anti-cancer cell proliferation properties of GpM cell suspension culture were significantly higher than those of natural plants’ extracts. In this framework, GpM in cell suspension cultures was found to inhibit the proliferation of several human cancer cells. Biomass accumulation of GpM in cell suspension cultures may contribute to the development of efficient strategies for plant-derived anticancer compound production.

Establishment of callus and cell suspension cultures of Ariocarpus retusus (Scheidweiler)

Ariocarpus retusus is an endemic species of Mexico subject to special protection by the Mexican government due to its vulnerability to habitat disturbance. Previous studies have reported secondary metabolites in A. retusus with interesting biological activity. However, the use of these secondary metabolites has limitations. The aim of this study was to establish protocols for obtaining friable callus and cell suspension cultures that allow the subsequent development of strategies for obtaining secondary metabolites. Seedlings were obtained from seeds which were exposed to different concentrations of auxins and cytokinins to induce callogenesis. The callus generated was subcultured in Murashige and Skoog (MS) medium with modifications in the content of ammonium nitrate and potassium nitrate, and incubated in different photoperiod conditions (16:8 h light: dark and continuous darkness). Likewise, cell suspensions were established and characterized by friable callus. The MS medium supplemented with 2 mg/L 2,4-D, 2 mg/L BAP, and 3 mg/L KIN allowed the development of vigorous callus. Callus friability was enhanced by decreasing ammonium nitrate (410 mg/L) and increasing potassium nitrate (2850 mg/L). The absence of light during incubation induced friable callus. The addition of myo-inositol (3 mg/L) significantly (p = 0.000) influenced the increase in biomass of cell suspensions.

Fruit derived callus and cell suspension culture as promising alternative sources for mogrosides production in Siraitia grosvenorii (Swingle) C. Jeffrey: a zero-caloric natural sweetener

The fruit of Siraitia grosvenorii (Luo Han Guo) is commercially consumed for its zero-calorie natural sweetener compounds known as mogrosides. The present work aimed to establish a cell culture platform from the fruit mesocarp and provide an alternative source for mogrosides production in S. grosvenorii. Bavistin at 1.0% with mercuric chloride at 0.1% were effective concentrations for explants sterilization. The maximum frequency of callus induction was observed in mesocarp (95.78%) and stem (97%) explants cultured on Murashige and Skoog (MS) medium supplemented with thidiazuron (0.5 mg/L) and picloram (2.5 mg/L). The maximum proliferation response of suspended cells in suspension culture was observed in MS medium fortified with thidiazuron (0.3 mg/L) and indole-3-butyric acid (0.3 mg/L). Mogroside V (2.96 mg/g DW), 11-oxo-mogroside V (0.66 mg/g DW), siamenoside I (0.26 mg/g DW), and mogroside IIIE (0.08 mg/g DW) were quantified in mesocarp callus using UPLC–PDA–ESI-Q-TOF-MS technique. A comparable content of mogroside V (1.76 mg/g DW) and 11-oxo-mogroside V (0.68 mg/g DW) was also quantified in cell suspension derived from mesocarp callus with enhanced cell biomass (FW; 184.58 g/L). Therefore, the in vitro culture derived from fruit mesocarp was shown to be a potential source for mogrosides production.

Enhanced production of Bacopa saponins by repeated batch strategy in bioreactor.

Cultivation of cell suspension culture of Bacopa monnieri targeting the production of bacosides was explored in a 5-l stirred tank reactor using statistically optimized conditions. The bioreactor cultivation conditions were modified and this led to profuse biomass growth (2.81 ± 0.20g/l) and total bacosides (1.26 ± 0.23mg/g in cells and 0.60 ± 0.11mg/l in fermenter broth) production in 9days. The values of static volumetric mass transfer coefficient (kLa), dimensionless mixing time (Nm) were measured in the bioreactor. The culture grew efficiently and produced enhanced amount of bacoside A (5.59 ± 0.41mg/g total bacosides in cells and 3.12 ± 0.13mg/l in the fermenter broth) using one cycle of repeated batch strategy adopted in the bioreactor for 15days. The intracellular concentration of bacoside A3 (1.18 ± 0.11mg/g), bacopaside II (2.09 ± 0.35mg/g), bacopaside X (0.79 ± 0.17mg/g) and bacopasaponin C (2.24 ± 0.23mg/g) were significantly higher in repeated batch as compared to batch bioreactor cultivation. The yield of total bacosides in the fermenter broth was 5-times higher in repeated batch as compared to batch cultivation. This strategy can be helpful for the enhanced production of other valuable triterpenoid saponins.

A novel spray bioreactor for the proliferation of plant callus; Hyoscyamus niger and Arnebia pulchra.

Unlike plant cell suspension culture, the proliferation of callus in bioreactors has received inadequate attention. The magnificent potential of plant callus becomes more appreciated as the research unfolds and promises interesting applications including the production of valuable metabolites, therapeutic antibodies, bioactive extracts with regenerating effects, and the generation of genetically improved plants. Issues such as the lack of 3D-access of the cells to the nutrients, using an interfering gelling substance as the support matrix, and the changes in the medium formulation during the growth phase were discouraging factors for extending research on this topic. Considering the existing drawbacks, a novel open-flow spray bioreactor (OFSB) was configured to circumvent the associated problems with the solid cell culture and promote the applicability of plant callus culture via improving the feeding strategy. Applying similar subculture conditions, the proliferation of Arnebia pulchra and Hyoscyamus niger calli as the examples of two important plant families (Boraginaceae and Solanaceae) was studied in the OFSB in comparison with similar calli that grew in Petri dishes and jars. A. pulchra and H. niger calli obtained the weight gains of (%87.3 and %106.7) in the Petri dishes, (%208.7 and %226) in the jars, and (%288.6 and %320.0) in OFSB, respectively, while no significant changes were observed in the productivity indices of the examined calli. The simple design of OFSB bypasses most of the notorious problems associated with solid plant callus culture. OFSB technical features allow the bioreactor to be used for growth optimization of various types of plant calli in a cost-effective manner.

Plant-Derived Cell-Free Biofactories for the Production of Secondary Metabolites.

Cell-free expression systems enable the production of proteins and metabolites within a few hours or days. Removing the cellular context while maintaining the protein biosynthesis apparatus provides an open system that allows metabolic pathways to be installed and optimized by expressing different numbers and combinations of enzymes. This facilitates the synthesis of secondary metabolites that are difficult to produce in cell-based systems because they are toxic to the host cell or immediately converted into downstream products. Recently, we developed a cell-free lysate derived from tobacco BY-2 cell suspension cultures for the production of recombinant proteins. This system is remarkably productive, achieving yields of up to 3 mg/mL in a one-pot in vitro transcription–translation reaction and contains highly active energy and cofactor regeneration pathways. Here, we demonstrate for the first time that the BY-2 cell-free lysate also allows the efficient production of several classes of secondary metabolites. As case studies, we synthesized lycopene, indigoidine, betanin, and betaxanthins, which are useful in the food, cosmetic, textile, and pharmaceutical industries. Production was achieved by the co-expression of up to three metabolic enzymes. For all four products, we achieved medium to high yields. However, the yield of betanin (555 μg/mL) was outstanding, exceeding the level reported in yeast cells by a factor of more than 30. Our results show that the BY-2 cell-free lysate is suitable not only for the verification and optimization of metabolic pathways, but also for the efficient production of small to medium quantities of secondary metabolites.

Вплив нейротрофічних факторів на популяцію серотонінергічних нейронів n.raphe в умовах культивування

Актуальність. Молекулярні механізми спеціалізації серотонінергічних нейронів зі стовбурових і прогеніторних нервових клітин вивчені далеко не повністю. Мета дослідження — визначення найбільш ефективних нейроіндукторів, що підтримують життєздатність та функціональну активність серотонінергічних нейронів та їх детермінованих попередників. Як критерії оцінки були обрані: експресія відповідних генів, загальний вміст серотоніну та чисельність серотонінергічних нейронів, візуалізованих за допомогою гістохімічної реакції. Матеріали та методи. Робота була виконана на суспензійній культурі клітин зони n.raphe з використанням гістохімічних методів, імуноферментного аналізу та полімеразної ланцюгової реакції. Результати. Установлено, що BDNF та NGF стимулюють збільшення чисельності серотонінергічних нейронів у дисоційованій культурі в 1,6 та 2 рази порівняно з контрольними зразками. Додавання в культуральне середовище BDNF та ретиноєвої кислоти призводить до зростання вмісту серотоніну в культуральному матеріалі. Експресія гена Nkx 2.2, що є маркером мітотично-активних попередників серотонінергічних нейронів, підтримується за наявності BDNF і збільшується під впливом EGF та FGFb. Експресія гена Pet1, що свідчить про наявність детермінованих прогеніторів серотонінергічних нейронів, підтримується всіма досліджуваними факторами, за виключенням екзогенного серотоніну. Експресія гена Tph1, маркера початку синтезу серотоніну, спостерігається під впливом FGFb, EGF та NGF. Висновки. Таким чином, наші дані показали, що вищевказані нейроіндуктори впливають на різні фази серотонінгенезу і можуть бути використані для його регуляції.

Statistical experimental designs to optimize the transient transfection of HEK 293T cells and determine a transfer criterion from adherent cells to larger-scale cell suspension cultures

The transient transfection of mammalian cells is a rapid and versatile platform for the manufacture of recombinant proteins, but industrial processes depend on reliable scalability and efficient conversion from adherent to suspension cell cultures. Here we describe the optimized transfection of HEK 293T cells in both culture formats. DMEM was the best transfection medium for adherent HEK 293T cells, so we determined the kinetics of linear polyethyleneimine (LPEI) polyplex formation with plasmid DNA (pDNA) and subsequent cellular uptake. Statistical experimental designs revealed optimal transfection efficiency using 0.7 pg pDNA and 4.5 pg LPEI per cell. We used the amount of pDNA and LPEI per cell as the transfer criterion for HEK 293T/17 SF cell suspension cultures in FreeStyle 293 medium and confirmed optimal transfection at 1.1 pg pDNA and 6.6 pg LPEI per cell. We observed a strong correlation between polyplex size, transfection efficiency and post-transfection cell viability. Suspension cell transfection could be scaled to a 100-mL working volume without loss of efficiency. We conclude that pg pDNA and pg LPEI per cell is a suitable transfer criterion allowing the optimization of transient transfection using statistical experimental designs, thus minimizing the amount of pDNA and LPEI used without sacrificing transfection efficiency.

Effect οf elicitοrs οn cell suspensiοn culture οf Urginea maritima L. tοwards prοductiοn οf prοscillaridin A.

Urginea maritima (L.f.) Baker (Hyacinthaceae) is a perennial bulbous medicinal plant that is currently at risk of extinction. Squill (white sea onion) is an analogous cardiotonic to digitalis. The purpose of the current work was to assess the optimal growth conditions for Urginea cells to synthesize the cardiac glycoside proscillaridin A by involving illumination, carbon source, methyl jasmonate (MJ), and culture system. When cells were cultured for 28 days at 21 ± 2 ∘C in the dark on Murashige and Skoog (MS) media containing 1 mg/L 2, 4-D and 0.5 mg/L Kin and 30 g/L sucrose, the cell proliferation, and proscillaridin A synthesis were effectively controlled. At low concentrations, MJ stimulated the synthesis of proscillaridin A (PsA). High-performance liquid chromatography (HPLC) analysis of suspension extracts demonstrated that the callus maintained in MS media enriched with 1 mg/L 2, 4-D and 0.5 mg/L Kin yielded a greater formation of Proscillaridin A (141.31 mg/g DW) than untreated plants. The results indicate that in vitro cultures of U. maritima may be an excellent source of proscillaridin A. Moreover, it is one of the most important cardiac glycoside, which has been found to exhibit anticancer activities. Suspension cultures of Urginea cells could be as highly productive as a callus culture.

Untargeted metabolomics analysis reveals the elicitation of important secondary metabolites upon treatment with various metal and metal oxide nanoparticles in Hypericum perforatum L. cell suspension cultures

In this study, the effects of treatment with Ag, Au, Cu, Pd, CeO2, CuO, TiO2 and ZnO nanoparticles (NPs) on plant secondary metabolism were tested using an untargeted metabolomic approach in cell suspension cultures of Hypericum perforatum L. Variations in the accumulation of secondary metabolites were observed in cultures after exposure to NPs. Overall, the number of compounds increased by metal NPs was higher than that of metal oxides. Among the metal and metal oxide NPs tested, Ag and CuO respectively induced the greatest changes in secondary metabolism. Ag NPs induced the cellular accumulation of bisxanthone (540.3 fold) gancaonin O (214.2 fold) and fusaroskyrin (98.6 fold). Compounds that were most induced by other NPs were: hyperxanthone C (Au), apigenin (Cu), emodin (Pd), emodin anthrone (CeO2), dihydroxydimethoxyxanthone I (CuO), quercetin (TiO2) and gallic acid (ZnO). The presented results show that the secondary metabolites elicited in H. perforatum vary between the types of NPs.

Enhanced production of pinosylvin stilbene with aging of Pinus strobus callus and nematicidal activity of callus extracts against pinewood nematodes

Pinosylvin stilbenes are phenolic compounds mainly occurring in the Pinaceae family. We previously reported that the accumulation of two pinosylvin stilbene compounds, dihydropinosylvin methyl ether (DPME) and pinosylvin monomethyl ether (PME), in Pinus strobus trees was highly enhanced by infection with pine wood nematodes (PWNs: Bursaphelenchus xylophilus), and these two compounds showed strong nematicidal activity against PWNs. In this work, we established a system of pinosylvin stilbene (DPME and PME) production via the in vitro culture of P. strobus calli, and we examined the nematicidal activity of callus extracts. Calli were induced from the culture of mature zygotic embryos of P. strobus. Optimized growth of calli was obtained in 1/2 Litvay medium with 1.0 mg/L 2,4-D and 0.5 mg/L BA. DPME and PME accumulation did not occur in nonaged (one-month-old) calli but increased greatly with prolonged callus culture. The concentrations of DPME and PME in three-month-old dark-brown calli were 6.4 mg/g DW and 0.28 mg/g DW, respectively. The effect of methyl jasmonate treatment on the accumulation of DPME and PME was evaluated in cell suspension culture of P. strobus. However, the treatment appeared to show slight increase of DPME accumulation compared to callus browning. A test solution prepared from crude ethanol extracts from aged calli (three months old) containing 120 µg/ml DPME and 5.16 µg/ml PME treated with PWNs resulted in 100% immobilization of the adult PWNs and 66.7% immobilization of the juvenile PWNs within 24 h. However, nonaged callus extracts did not show any nematicidal activity against juvenile PWNs and showed less than 20% nematicidal activity against adult PWNs. These results indicate that pinosylvin stilbenes can be effectively produced by prolonged culture of P. strobus calli, can be isolated using simple ethanolic extraction, and are applicable as beneficial eco-friendly compounds with nematicidal activity against PWNs.

Characterization of lipid droplets from a Taxus media cell suspension and their potential involvement in trafficking and secretion of paclitaxel.

Our paper describes the potential roles of lipid droplets of Taxus media cell suspension in the biosynthesis and secretion of paclitaxel and, therefore, highlights their involvement in improving its production. Paclitaxel (PTX) is a highly potent anticancer drug that is mainly produced using Taxus sp. cell suspension cultures. The main purpose of the current study is to characterize cellular LDs from T. media cell suspension with a particular focus on the biological connection of their associated proteins, the caleosins (CLOs), with the biosynthesis and secretion of PTX. A pure LD fraction obtained from T. media cells and characterized in terms of their proteome. Interestingly, the cellular LD in T. media sequester the PTX. This was confirmed in vitro, where about 96% of PTX (C0PTX,aq [mgL-1]) in the aqueous solution was partitioned into the isolated LDs. Furthermore, silencing of CLO-encoding genes in the T. media cells led to a net decrease in the number and size of LDs. This coincided with a significant reduction in expression levels of TXS, DBAT and DBTNBT, key genes in the PTX biosynthesis pathway. Subsequently, the biosynthesis of PTX was declined in cell culture. In contrast, treatment of cells with 13-hydroperoxide C18:3, a substrate of the peroxygenase activity, induced the expression of CLOs, and, therefore, the accumulation of cellular LDs in the T. media cells cultures, thus increasing the PTX secretion. The accumulation of stable LDs is critically important for effective secretion of PTX. This is modulated by the expression of caleosins, a class of LD-associated proteins with a dual role conferring the structural stability of LDs as well as regulating lipidic bioactive metabolites via their enzymatic activity, thus enhancing the biosynthesis of PTX.

Rosmarinic acid production in cell suspension cultures of Ehretia asperula Zollinger & Moritz

Plant cell cultures provide an alternative means for producing secondary compounds in food, cosmetic and pharmaceutical industries. Ehretia asperula Zollinger & Moritzi is used as a traditional medicine for the treatment of liver detoxification, ulcers, tumors, inflammation and enhancing the body's resistance in Vietnam. The study was carried out to select suitable callus line for cell suspension cultures of E. asperula Zollinger & Moritzi and investigate the effects of inoculum size, rotation speed and naphthalene acetic acid (NAA) on the proliferation of cell suspension cultures. In addition, the influence of light intensity on the growth and rosmarinic acid (RA) biosynthesis of cell suspension was also surveyed. After 4 weeks of culture, the white to pale yellow friable callus expanded significantly with a fresh weight (FW) of 0.788 g and a high RA content of 2.062 mg/g FW. An appropriate medium for cell proliferation was the liquid B5 medium, which contained 30 g/l glucose, 0.1 mg/l benzyl adenine (BA) and 0.4 mg/l NAA. The results also demonstrated that a 1:20 ratio (w/v) inoculum size, darkness and rotation speed of 90 rpm were the optimal conditions for the proliferation and RA accumulation to 188.217 mg/l in 4 weeks of culture. These findings showed that E. asperula Zollinger & Moritzi cell suspension cultures could be a potential alternative approach for RA production in vitro.

Establishment of cell suspension cultures of Azadirachta indica for the production of nimbolide

The present study aimed to establish an effective cell suspension medium for the production of nimbolide from the callus culture derived from leaf explants of Azadirachta indica. The callus was induced in MS basal medium supplemented with different concentrations of auxins and cytokinins and the highest callusing percentage (93.3%) was obtained in MS basal medium supplemented with 1.0 mgL–1 IBA and 0.5 mgL–1 BAP. The friable calli were transferred to suspension cultures that were prepared with full strength and half strength liquid MS medium with varying concentrations of IBA and BAP. HPLC analysis revealed the presence of nimbolide in all the cell suspension culture and higher content of about 0.04% was measured in half MS medium supplemented with 1.0 mgL−1 IBA and 0.5 mgL−1 BAP. Thus, this study has identified optimum culture conditions that favours higher amount of nimbolide which has several applications both in agriculture and healthcare industry.

High-Throughput Transfection and Analysis of Soybean (Glycine max ) Protoplasts.

With the advent of plant synthetic biology, there is an urgent need to develop plant-based systems that are able to effectively enhance the speed of design-build-test cycles to screen large numbers of synthetic constructs. Thus far, protoplasts have served to fill this need, with cell suspension cultures serving as the primary source tissue to enable high-throughput protoplast experimentation. The possibility to use low-cost food-grade enzymes for cell wall digestion along with polyethylene glycol (PEG)-mediated transfection makes protoplasts particularly suited to automation and high-throughput screening. In other systems for which synthetic biology is well established (model bacteria and yeast), libraries of components, i.e., promoters, 5' untranslated regions, 3' untranslated regions, terminators, and transcription factors, serve as the basis for the design of complex genetic circuits. In order for synthetic biology to make similar strides in plant biology, well-characterized libraries of functional genetic parts for plants are required, necessitating the need for high-throughput protoplast assays.In this chapter, we describe an optimized method for the preparation of soybean (Glycine max ) dark-grown cell suspension cultures, followed by protoplast isolation, automated transfection , and subsequent screening.

Recombinant Protein Production in Plants: A Brief Overview of Strengths and Challenges.

The first recombinant proteins were produced in microbes and animal cells cultivated in bioreactors. These systems have become the standard for industrial-scale recombinant protein manufacturing. Later, the production of recombinant proteins was demonstrated in whole plants, which differ morphologically from cell-based systems and require completely different cultivation conditions. Over time, additional plant-based production platforms were established, including hairy roots and cell suspension cultures, which are more similar to conventional cell-based systems in terms of morphology, procedures, and equipment requirements. In this brief overview of the field, we explain why plant-based systems are becoming increasingly attractive for the production of valuable proteins with scientific and commercial applications, but also highlight the challenges that these systems must overcome to achieve more widespread industrial utilization. We discuss various laboratory protocols and approaches for the production of recombinant proteins in plants, as well as strategies to optimize yields, and the regulatory and legal framework.

Effect of basal medium on growth and polyphenols accumulation by Gardenia jasminoides Ellis cell suspension

The goal of this study was to analyze the effect of different medium bases on accumulation of biomass and secondary metabolites production by Gardenia jasminoides Ellis. cell suspension culture. The result revealed that different media have significant effect on biomass accumulation and production of polyphenol compounds. HPLC analyses showed that the higher polyphenol content was achieved when the cells were cultivated on McCOWN Woody plant Medium (WP) media for 14 days on darkness, at 24°C. At this conditions, the cells produced maximal amounts of catechin (24.44±2.46 µg/g DW), chlorogenic acid (20.26 ±3.68 µg/g DW), epicatechin (70.44±7.46 µg/g DW) and rutin (45.90±0.26 µg/g DW). Both MS and WP media were optimal for biomass accumulation. When cultivated on these media, Gardenia cells accumulated maximal amount of dry biomass ADB = 0.99±0.1g/100ml and ADB = 0.93±0.04 g/100ml for МS and WP, respectively. The corresponding growth indexes were GIDW = 1.68±0.2 on MS and GIDW = 1.58±0.07 on WP medium. The reported results are the first step of future optimization of the nutrient medium composition and cultivation conditions essential for the scale -up of cultivation process of G. jasminoides cell suspension culture for mass production of polyphenolic compounds.

Banana somatic embryogenesis and biotechnological application

As one of the most important economic crop for both staple food and fruit widely cultivated in tropics and subtropics, banana (<italic>Musa</italic> spp.) is susceptible to a plethora of abiotic and biotic stresses. Breeding cultivars resistant to abiotic and biotic stressors without adverse effects on yield and fruit quality are the objectives of banana improvement programs. However, conventional breeding approaches are time-consuming and severely hampered by inherent banana problems (polyploidy and sterility). Therefore, genetic transformation is becoming increasingly popular and can provide rapid solutions. Numerous efforts have been made to develop superior banana cultivars with better resistance to abiotic and biotic stresses and optimum yields using genetic modification strategies. Somatic embryogenesis (SE) through embryogenic cell suspension (ECS) cultures is an ideal recipient system for genetic transformation in banana. The purpose of this paper is to review the current status of banana somatic embryo research, clarify the way of banana somatic embryo induction and culture, and summarize the main influencing factors in the process of somatic embryogenesis. At the same time, their applications in breeding technologies such as cryopreservation, protoplast culture, genetic transformation and gene editing were also summarized, in order to provide reference for the research and practical application of banana somatic embryogenesis in the future.