Plant cell and organ cultures have emerged as potential sources of secondary metabolites, which are used as pharmaceuticals, agrochemicals, flavors, fragrances, coloring agents, biopesticides, and food additives. In recent years, various strategies have been developed to assess biomass accumulation and synthesis of secondary compounds in cultures. Biomass accumulation and metabolite biosynthesis are two-stage events, and the parameters that control the growth and multiplication of cultured cells/organs and biomass accumulation are controlled in the first stage. Parameters that assist with the biosynthesis of metabolites are controlled in the second stage. The selection of high-producing cells or organ clones; optimization of medium parameters such as suitable medium, salt, sugar, nitrogen, phosphate, and plant growth regulator levels; and physical factors such as temperature, illumination, light quality, medium pH, agitation, aeration, and environmental gas (e.g., oxygen, carbon dioxide, and ethylene) are controlled in the first stage of the culture process. Elicitation, replenishment of nutrient and precursor feeding, permeabilization, and immobilization strategies assist with the accumulation of metabolites and can be applied in the second stage of the culture process. By following stage-specific strategies, it is possible to produce large amounts of biomass with an increase in the accumulation of secondary compounds.

Production of secondary metabolites from cell and organ cultures: strategies and approaches for biomass improvement and metabolite accumulation

Automation of micropropagation via organogenesis or somatic embryogenesis in a bioreactor has been advanced as a possible way of reducing costs. Micropropagation by conventional techniques is typically a labour-intensive means of clonal propagation. The paper describes lower cost and less labour-intensive clonal propagation through the use of modified air-lift, bubble column, bioreactors (a balloon-type bubble bioreactor), together with temporary immersion systems for the propagation of shoots, bud-clusters and somatic embryos. Propagation of Anoectochilus, apple, Chrysanthemum, garlic, ginseng, grape, Lilium, Phalaenopsis and potato is described. In this chapter, features of bioreactors and bioreactor process design specifically for automated mass propagation of several plant crops are described, and recent research aimed at maximizing automation of the bioreactor production process is highlighted.

Application of bioreactor systems for large scale production of horticultural and medicinal plants

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/j.febslet.2006.08.044

Dammarenediol-II synthase, the first dedicated enzyme for ginsenoside biosynthesis, in Panax ginseng

The invention provides methods and compositions useful for the production of oil, fuels, oleochemicals, and other compounds in microorgansims. In particular, the invention provides oil-bearing microorganisms and methods of low cost cultivation of such microorganisms. The invention also provides microbial cells containing exogenous genes encoding, for example, a lipase, sucrose transporter, sucrose invertase, fructokinase, a polysaccharide-degrading enzyme, fatty acyl-ACP thioesterase, fatty acyl-CoA/aldehyde reductase, fatty acyl-CoA reductase, fatty aldehyde reductase, fatty aldehyde decarbonylase, and/or an acyl carrier protein. The invention also includes methods of manufacturing transportation fuels such as renewable diesel, biodiesel, and renewable jet fuel.

Production of oil in microorganisms

Biotechnology is playing a vital alternative role in the production of pharmaceutical plant secondary metabolites to support industrial production and mitigate over-exploitation of natural sources. High-value pharmaceuticals that include alkaloids, flavonoids, terpenes, steroids, among others, are biosynthesized as a defensive strategy by plants in response to perturbations under natural environmental conditions. However, they can also be produced using plant cell, tissue, and organ culture techniques through the application of various in vitro approaches and strategies. In the past decades, efforts were on the clonal propagation, biomass and secondary metabolites production in the in vitro cultures of medicinally important plants that produce these molecules. In recent years, the effort has shifted towards optimizing culture conditions for their production through the application of cell line selection, elicitation, precursor feeding, two-phase co-culture among cell, tissue, and organ culture approaches. The efforts are made with the possibility to scale-up the production, meet pharmaceutical industry demand and conserve natural sources of the molecules. Applications of metabolic engineering and production from endophytes are also getting increasing attention but, the approaches are far from practical application in their industrial production.

Secondary metabolism of pharmaceuticals in the plant in vitro cultures: strategies, approaches, and limitations to achieving higher yield

A pilot-scale culture of multiple adventitious roots of ginseng was established using a balloon-type bubble bioreactor. Adventitious roots (2 cm) induced from callus were cultured in plastic Petri dishes having 20 ml of solid Schenk and Hildebrandt (1972) medium containing 3% sucrose, 0.15% gelrite, and 24.6 μM indole-3-butric acid. An average of 29 secondary multiple adventitious roots were produced after 4 weeks of culture. These secondary roots were elongated on the same medium, reaching a length of 5 cm after 6 weeks of culture. A time course study revealed that maximum yields in 5-l and 20-l bioreactors were approximately 500 g and 2.2 kg at day 42 with 60 g and 240 g inoculations, respectively. Cutting twice during the culture increased the total amount of biomass produced. The root biomass in a 20-l balloon-type bubble bioreactor was 2.8 kg at harvest with 240 g of inoculum after 8 weeks of culture. The total saponin content obtained from small-scale and pilot-scale balloon type bubble bioreactors was around 1% based on dry weight. Inoculation of 500 g fresh weight of multiple adventitious roots into a 500 l balloon-type bubble bioreactor with cutting at 4 and 6 weeks after inoculation produced approximately 74.8 kg of multiple roots. The ginsengnoside profiles of these multiple adventitious roots were similar to profiles of field-grown ginseng roots when analyzed by HPLC.

Pilot-scale culture of adventitious roots of ginseng in a bioreactor system

Among the various plant growth regulators (PGR), 9.0 μM 2,4-D(2,4-dichlorophenoxyacetic acid) turned out to be the best for callogenesis and 4.5 μM 2,4-D was revealed to be more suitable for callus proliferation. Of the four basal media, the highest number of 8.2 adventitious roots was induced in Woody plant medium (WPM) containing 14.8 μM IBA(indole 3-butyic acid). F tests on each auxin treatment in the ANOVA revealed highly significant difference at α=0.01. The number of multiple adventitious roots (MARs) formed in each adventitious roots highly depended on the types and levels of auxin tested. At both 24.6 μM and 34.4 μM IBA, more than 25 MARs were induced after 4 weeks of culture. When different medium were applied, immersion type showed the highest fresh weight of MARs at the time of harvest. With 160 g fresh weight of inoculum, about 14.2 times of biomass was obtained after 6 weeks of culture. Identical fractionation pattern of the amplified fragment length polymorphism (AFLP) analysis was observed between the adventitious root and donor of a mountain ginseng. This observation suggested that any rearrangements of the genomic DNA had not occurred in the adventitious roots after 1 year of cultures. The cultivated ginseng and the mountain ginseng groups could be distinguished by 2 AFLP markers.

Induction and cultures of mountain ginseng adventitious roots and AFLP analysis for identifying mountain ginseng

Large-scale Culture of Plant Cell and Tissue by Bioreactor System

Calli were induced from 300,000 embryos isolated from immature to mature stage of seeds collected on late September from 14 elite trees. When the embryos were cultured onto plastic Petri-dish containing 20 mL of modified B5 basal medium supplemented with 3% (w/v) sucrose, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.5% (w/v) polyvinyl polypyrrolidon (PVPP), 2×MS vitamins, 0.5 mg/L gibberellic acid, and 10 mg/L 2,4-D after 2 weeks of culture, yellowish-white calli were immediately formed on the surfaces of embryos, and subcultured for 4 weeks in same culture medium. Because most of calli maintained for more than 3 months were revealed differences in their colors, surface texture, and growth rate, visual selection was made for first round screening. When the size of visually selected calli larger than 19 mm in their diameter were inoculated, persistent proliferation was observed. Among the plating methods tested for the selection of rapid growing cell lines at single cell and/or small cell aggregate level, 2-layer spread plating revealed as the best for single cell cloning. To enhance cell growth and maintain high rate of viability for long-term culture of yew cells in bioreactor, final cell volume less than 50% in SCV seemed to be the best. Time course study revealed that 30% of inoculum density was suitable for fed batch culture. Among the tested conditional media, the rate of 1∶2 (old medium: fresh medium) was recorded at the best for cell growth.

Sung Mee Choi

Papers

Pilot-scale culture of adventitious roots of ginseng in a bioreactor system

Induction and cultures of mountain ginseng adventitious roots and AFLP analysis for identifying mountain ginseng

Large-scale Culture of Plant Cell and Tissue by Bioreactor System

Selection and proliferation of rapid growing cell lines from embryo derived cell cultures of yew tree (Taxus cuspidata Sieb. et Zucc)