Plant tissue culture, regeneration and secondary metabolites

Totipotency is the ability of a single living plant cell to give rise to a whole fertile plant, because it carries the complete genome and can reprogram which genes it expresses. Most living, nucleated plant cells keep this ability after differentiation, unlike most differentiated animal cells. Cells that lose their nucleus or die at maturity (sieve tube elements, xylem vessels) cannot regenerate a plant.

Regeneration in culture is a series of cell fate transitions. A differentiated cell of the explant first dedifferentiates, losing its specialised identity and resuming division. It then becomes competent (able to respond to a signal). Under a suitable hormone signal it becomes determined to a new fate, and it finally redifferentiates into a shoot, a root, or an embryo. Wounding of the explant and auxin are the usual triggers for the first step.

An explant is the piece of plant (leaf disc, node, shoot tip, anther, cotyledon) placed in culture. Young, actively growing tissue and meristems respond best. Explants are surface sterilised (for example 70% ethanol for 30 to 60 s, then sodium hypochlorite or mercuric chloride, then several rinses in sterile water). Media, vessels and tools are autoclaved at 121 °C and 15 psi for 15 to 20 min, and work is done in a laminar air-flow cabinet. Heat-labile components (gibberellic acid, some vitamins, antibiotics) are filter sterilised through 0.22 µm filters and added after autoclaving.

MS medium contains macronutrients (nitrate and ammonium nitrogen, potassium, calcium, magnesium, phosphate, sulfate), micronutrients, Fe-EDTA, vitamins (myo-inositol, thiamine and others), a carbon source (sucrose, usually 20 to 30 g/L) and plant growth regulators. It is solidified with agar (about 0.6 to 0.8% w/v) or used liquid for suspensions. The pH is adjusted to about 5.6 to 5.8 before autoclaving. Browning from phenolic oxidation is reduced with activated charcoal, polyvinylpyrrolidone (PVP) or ascorbic acid.

The auxin-to-cytokinin ratio steers organogenesis. A high auxin to cytokinin ratio favours root formation, a high cytokinin to auxin ratio favours shoot formation, and a roughly balanced ratio favours undifferentiated callus growth. This is a general rule, and the absolute concentrations and the species matter.

Organogenesis forms shoots or roots (unipolar structures that stay connected to the vascular tissue of the explant). It is direct when organs arise straight from the explant with no intervening callus, and indirect when the explant first forms callus and organs arise from the callus. Indirect routes are more likely to give genetic variation. Regeneration through callus usually runs in stages on different media: a callus induction medium (relatively high auxin), then a shoot induction medium (higher cytokinin), then a rooting medium (an auxin such as IBA or NAA).

Somatic embryogenesis forms bipolar embryos (shoot and root poles together) from somatic cells, with no vascular connection to the parent tissue. This is the key difference from organogenesis. It is direct when embryos form from explant cells that are already embryogenically determined, and indirect when a callus phase is needed to induce cells to become embryogenic. In dicots the stages are globular, heart, torpedo and cotyledonary. In monocots they are globular, scutellar and coleoptilar. Induction often uses 2,4-D, and embryo development usually needs the auxin removed or reduced. ABA helps maturation.

Meristem (shoot-tip) culture for virus-free plants. The apical dome with one or two leaf primordia (about 0.1 to 0.5 mm) often carries little or no virus, because it has no differentiated vascular tissue and its cells divide rapidly. Culturing this tip gives virus-free plants, often after heat treatment of the stock plant (thermotherapy) or with antiviral chemicals. The plants are then tested for virus (for example by ELISA or PCR). The method is used for potato, banana, sugarcane and many ornamentals.

Haploid plants are obtained by culturing anthers or isolated microspores (androgenesis), which regenerate from the haploid microspore cells either through embryos or callus. Culture of unfertilised ovaries or ovules (gynogenesis) is another route. Anther culture can also regenerate diploid plants from the anther wall, so the ploidy of regenerated plants is checked. Treating haploids with colchicine doubles the chromosome number to give doubled haploids, which are completely homozygous in one generation. This shortens the time to get pure lines.

Artificial (synthetic) seeds are somatic embryos, shoot tips or axillary buds encapsulated in a protective, nutrient-containing gel. The usual matrix is sodium alginate (about 2 to 3%), which is dropped into a calcium chloride solution (about 50 to 100 mM). Calcium ions replace sodium and form calcium alginate beads. The bead may hold nutrients, growth regulators and antimicrobial agents. Synthetic seeds are hydrated (encapsulated in a hydrogel such as calcium alginate) or desiccated (hardened somatic embryos, dried, with or without a coating). The advantages are easy handling and storage, clonal uniformity, and large-scale propagation. The limits are poor conversion to plants, lack of natural dormancy, and sensitivity to desiccation.

A callus culture is maintained on solid medium and subcultured every few weeks. A cell suspension is started by transferring a friable callus into liquid medium and shaking it (about 100 to 150 rpm), which breaks the callus into single cells and small aggregates. Suspensions are subcultured by diluting into fresh medium, and are easier to scale up in bioreactors, to sample, and to treat uniformly with nutrients or elicitors. Plant cells divide slowly: their doubling times are usually measured in days, not the minutes to hours typical of bacteria.

Measuring growth. Common measures are fresh weight, dry weight, cell number (haemocytometer), packed cell volume (PCV) after centrifugation or settling, culture viability (fluorescein diacetate stains live cells, Evans blue stains dead cells), and the electrical conductivity of the medium, which falls as ions are taken up.

Nutrient optimisation. Sucrose is mostly hydrolysed by cell-wall invertase into glucose and fructose, which the cells take up. Ammonium is usually taken up before nitrate. Ammonium uptake acidifies the medium and nitrate uptake makes it more alkaline, so the pH often first falls and then rises. Phosphate is often depleted early, and a low phosphate level can raise secondary metabolite output while limiting growth. Conditions are optimised by changing one factor at a time or, more efficiently, by statistical designs and response surface methods that test several factors together (sucrose, nitrogen source and ratio, phosphate, growth regulators, inoculum size, agitation, aeration, temperature, light).

Secondary metabolites are compounds not needed for basic growth but important for defence, signalling and attraction. The main classes are alkaloids (nitrogen-containing), terpenoids (built from isoprene units) and phenolics (phenylpropanoids, flavonoids). They are often made at low levels, only in specific tissues or developmental stages, and frequently in the non-growth (stationary) phase, so growth and production may be run in two stages: a growth medium, then a production medium.

Elicitors are molecules or treatments that trigger a defence response and so increase secondary metabolite synthesis. Biotic elicitors come from living sources (yeast extract, chitosan, fungal cell-wall fragments, pectin fragments called oligogalacturonides). Abiotic elicitors are chemical or physical (heavy metal salts, UV light, osmotic stress). The plant signal molecules methyl jasmonate and salicylic acid are classed differently in different sources; here they are grouped with the chemical (abiotic) elicitors. The response involves receptor perception, calcium influx, reactive oxygen species and signalling that up-regulates the biosynthetic genes. Concentration and exposure time must be optimised, because too strong an elicitor kills the cells.

Hairy root culture uses Agrobacterium rhizogenes (now also classified as Rhizobium rhizogenes), which carries a Ri (root-inducing) plasmid. Wound phenolics such as acetosyringone induce the vir genes of the plasmid, and the bacterium transfers T-DNA into the genome of the wounded plant cells. The T-DNA carries the rol genes (mainly rolA, rolB, rolC), which make the transformed cells form abundant roots, largely by raising their sensitivity to auxin. The T-DNA also directs the synthesis of opines. After co-cultivation the bacteria are killed with an antibiotic (for example cefotaxime) and roots are grown on hormone-free medium.

Hairy roots grow quickly in hormone-free medium and are stable, so they make root-type metabolites (for example tropane alkaloids) at levels often similar to or higher than the intact root. Their limits are shear damage, uneven oxygen and nutrient supply in dense root mats, and the need for special bioreactors (airlift, bubble column, mist or trickle-bed types).

Somaclonal variation is the genetic or epigenetic variation seen among plants regenerated from cells or tissues grown in culture, especially through a callus or suspension phase. Plants regenerated from gametic cells show the same effect, called gametoclonal variation.