Plant in vitro culture reports
A manuscript on producing bioactive compounds in plant cell, tissue or organ culture follows the usual research-article structure, but it has to give the botanical name of the plant, the type of culture and its conditions, any strategies used to raise productivity, the bioreactor and the extraction and analysis of the compounds in the detail the field expects, and it has to make its novelty plain to the journal that judges it.
The field and what it produces
Phytochemicals are divided by their function in plant metabolism into primary and secondary metabolites. Primary metabolites, such as carbohydrates, proteins and lipids, are directly involved in plant growth and development, while secondary metabolites, such as alkaloids, terpenes, flavonoids and steroids, are made in a defence response to threats and stresses in the natural environment and are valued in medicines, flavours, dyes, repellents and cosmetic ingredients. In 2023 market demand for secondary metabolites was still growing, which may endanger some medicinal plants through overharvesting, and chemical synthesis of compounds with complex structures is economically unfeasible. Plant cell, tissue and organ cultures had by then offered in vitro production of bioactive secondary metabolites for 60 years, and with demand for phytochemicals increasing, they are an environmentally sustainable method of producing them. In 2023 a PubMed search for "in vitro plant cultures" displayed more than 1000 publications a year over the recent decade, which indicates the potential for medical and scientific writers in the field.
Paclitaxel, a broad-spectrum anticancer diterpenoid discovered in the 1960s and marketed since 1993 as Taxol, is a key example of a product made commercially through plant in vitro culture, and in 2023 its annual world demand was 800–1000 kg. Isolating it directly from the inner bark of yew trees is ecologically unsustainable because its content is too low: six slow-growing, 100-year-old trees are needed to produce the 2 g of pure drug that treats one patient. Taxus cell suspension cultures proved an alternative route, and a Taxus chinensis suspension culture elicited with methyl jasmonate gave one of the highest paclitaxel yields reported by 2023, 565 mg per litre or 29.3 mg per gram of dry weight. In 2023 the production system developed by Phyton Biotech, based on cultivating T. chinensis in 75,000-litre bioreactors, was the largest commercially viable plant cell culture application. Other medicinally important compounds made in plant in vitro cultures include antitumour indole alkaloids from Catharanthus roseus, anticholinergic tropane alkaloids from Datura stramonium and Atropa belladonna, and immunomodulating ginsenosides from Panax ginseng. Research on these cultures has run since the mid-1960s, but by 2023 plant in vitro systems had not been broadly commercialised, and efforts continued to overcome the limits on scaling up secondary metabolite production, such as insufficient knowledge of biosynthetic pathways and the slow growth of the cultures.
Botanical names and Latin terms
Scientific plant names under the International Code of Nomenclature for algae, fungi, and plants are required in research on plants, because they ensure scientific accuracy and avoid citation errors. "Ginseng in vitro cultures" is not enough, because several species of the genus Panax are used to establish cultures that produce ginsenosides, P. ginseng most often but also P. quinquefolius and P. sikkimensis, and each gives a different ginsenoside yield. So the manuscript must give the precise botanical name of the explant source, preferably with the genetic identification of the mother plant and the place where its archived herbarium specimen, the representative voucher specimen, is stored, such as a university department or a horticultural library.
The Latin binomial system, first developed by Carl Linnaeus, has its own spelling rules. The full name is written in italics, with the genus capitalised and the species in lower case, and the name of the authority who named the plant, usually given at first mention, is not italicised, as in Mentha piperita L., where L. stands for Linnaeus. Once the full name has been used, the genus can be shortened to its initial where nothing can be confused, as in M. piperita, or the common name can be used, neither italicised nor capitalised, as in mint. Higher taxa such as families and orders are written in standard lettering, as in Lamiaceae, formerly Labiatae, the mint family. "Sp." after a genus refers to the genus in general, and "spp." to several species of that genus. Taxonomic changes leave one plant with several names, as Rhododendron tomentosum Harmaja was formerly Ledum palustre L., and using only an outdated name may make bibliographic searches miss the article, though the outdated name can still be given as a synonym (syn.), especially where taxonomists disagree or the older name is widely known.
The target journal's instructions for authors and style guide clarify many further points, and some submission guidelines do not insist on italicising common Latin terms such as in vitro and in vivo.
The type of culture
Depending on the explant and the culture medium, the culture is a cell culture (a cell suspension, protoplasts or gametic cells), undifferentiated callus, a differentiated tissue such as a meristem, or an organ culture of shoots, roots or zygotic embryos. Cell suspensions seem a promising source of phytochemicals because they grow fast, which makes scale-up to bioreactors easier, but some secondary metabolites, essential oils among them, need a degree of differentiation, since several biosynthetic pathways may be fully developed only in particular plant organs. The manuscript should describe in detail the explant source, the meristematic tissue of the mother plant whose continuously dividing cells start the culture, and the culture initiation, along with the stages of micropropagation where they apply.
A root culture should be described as adventitious or hairy. Hairy roots arise from infection with strains of Rhizobium rhizogenes (formerly Agrobacterium rhizogenes), a soil-borne Gram-negative bacterium, an infection that leads to the integration of a root-inducing plasmid into the plant genome. Unlike non-transformed adventitious roots developed from stems or leaves, transgenic hairy roots grow rapidly in culture media without added auxins, and they are genetically stable and give high secondary metabolite production.
Culture conditions
Plant in vitro cultures are incubated in controlled aseptic conditions, in closed flasks or jars spaced evenly in a growth room. External factors may affect photosynthesis and other biochemical processes and induce anatomical and physiological changes, so light (its intensity, spectrum and photoperiod), humidity and temperature should be reported. A basic nutrient medium should be referenced to its original formulation without its exact composition, and any modification, such as a change to the medium's acidity, should be explained. Media are commonly abbreviated, as MS for Murashige and Skoog and SH for Schenk and Hildebrandt, and so are plant growth regulators, as BAP for 6-benzylaminopurine and TDZ for thidiazuron. Cytokinins, auxins, gibberellins and other substances added to the medium should be expressed in μM. Precise reporting of the combinations of plant growth regulators and their concentrations is essential, because they strongly affect shoot regeneration, rooting and callus formation. The manuscript also needs to say whether the medium was liquid (for shaken and stationary cultures), solid (for stationary cultures) or semi-solid with support for the developing plants, as during micropropagation, because this influences biomass growth.
Strategies for improving productivity
Once high-yielding cultures have been selected and the culture conditions optimised, techniques may be applied to improve biomass and secondary metabolite production. They should be described in the materials and methods in enough detail, with technical specifications, experimental design and data collection, for the study to be repeated and its findings verified. The techniques that typically need describing are:
- elicitation, the use of biotic or abiotic compounds or factors to trigger the plant's defence response
- permeabilisation, the release of product from the biomass into the culture medium
- immobilisation, fixing the biomass in or on a supporting material or matrix
- precursor feeding, adding exogenous or endogenous compounds that the cultures convert into secondary metabolites through biosynthetic pathways
- metabolic engineering, introducing genes that encode specific metabolic enzymes into the plant
The bioreactor
The bioreactor is a fundamental tool for scaling up in vitro production of secondary metabolites. It transfers nutrients and gases in bulk and keeps micro-environmental conditions constant, which promotes rapid biomass growth, and its automated cultivation saves time and reduces labour costs. Bioreactors for plant cultures differ from those for microbial cultures, because plant organs, and plant cells, which are larger than bacterial cells at 10–100 μm against 0.5–5 μm long, can be mechanically damaged in stirred-tank bioreactors and by foam in bubble-aerated ones. Each bioreactor is selected for the type of plant culture, with efficient oxygen and nutrient supply, low shear stress, proper mixing and a suitable support system in view. Bioreactors come with many modifications, such as mist spray, temporary immersion, columns of different shapes, a mesh or basket, and various agitation options, so an accurate description is essential, preferably with a schematic drawing. A reference is enough for a commercially available system, such as RITA, that has not been modified.
Downstream processing
Manuscripts on secondary metabolite production in plant cultures often describe downstream processing as an integral part of the research. Extracting the bioactive compounds from the plant material and analysing them qualitatively and quantitatively are essential for screening cultures and for assessing how effective a large-scale system is. Yield is usually expressed in mg per gram of dry weight, less often in mg per gram of fresh weight, or in mg per litre for a cell suspension culture, and bioreactor productivity can also be given in grams per litre per day. The spelling of metabolite names should be checked carefully, especially long names based on chemical structure, and for a compound that is not widely known, giving its chemical formula may be considered. Assigning the products to their phytochemical group, such as essential oils, flavonoids or alkaloids, helps in discussing their potential biological activity.
Publishing in plant biotechnology
Research articles on plant biotechnology do not differ significantly from other papers with an IMRAD structure, but describing plant in vitro cultures carries many specific nuances, from botanical nomenclature through establishing the culture and developing a bioreactor system to extracting and analysing the bioactive compounds. Medical writers can find these articles tricky because they need knowledge at the intersection of botany, chemistry, pharmacy and biotechnology, and an inaccurate description of methods or inadequate data analysis risks rejection by the journal.
The difficulty in publishing in plant biotechnology is the relatively long time it takes to develop a high-yielding plant in vitro system, so meeting the expectation of a scientific article every year requires creativity in proposing new solutions and innovative strategies. A study recent in 2023 found that the main reasons journals in agriculture and plant biology rejected manuscripts were lack of novelty, flaws in methods or data interpretation, inadequate data analysis and poor critical scientific thinking. So a vital task for the medical writer is to emphasise the novelty of the research clearly and to set out the complexity of in vitro secondary metabolite production transparently.