Plant Biotechnology — Totipotency, Tissue and Cell Culture, Secondary Metabolites and Transgenic Plants

Elicitors are compounds that trigger a plant cell’s defence responses, and with them the synthesis of many secondary metabolites (phytoalexins, alkaloids, terpenoids, phenolics). Biotic elicitors come from organisms — fungal cell-wall fragments, chitosan, yeast extract, glucan oligosaccharides — and abiotic elicitors are physical or chemical stresses — heavy-metal ions, UV light, osmotic stress — together with the signalling molecules methyl jasmonate and salicylic acid, which act inside the defence pathways themselves. An elicitor is perceived by receptors, triggers ion fluxes, reactive oxygen and kinase cascades, and switches on the transcription of biosynthetic genes. Its effect depends strongly on dose, timing and exposure time: elicitation is usually applied late in the exponential phase, when there is enough biomass, because the defence response diverts resources from growth and can kill cells at high doses.

The growth regulators are not interchangeable with elicitors: a regulator drives division and morphogenesis along the paths the cell already uses for growth, whereas an elicitor drives the cell to make defensive secondary chemistry it would otherwise make only when attacked. A production protocol typically uses both — regulators to build the biomass, an elicitor near the end to switch that biomass over to the product.

Methodology. An explant (leaf disc, stem segment, embryo, meristem) is surface-sterilised — typically a brief rinse in 70% ethanol followed by sodium hypochlorite and repeated washes in sterile water — and placed on a medium, most often Murashige and Skoog (MS) medium (1962). MS supplies macronutrients (notably high nitrate and ammonium nitrogen), micronutrients, iron as Fe-EDTA, vitamins (thiamine, nicotinic acid, pyridoxine) and myo-inositol, sucrose (about 3%) as the carbon source because cultured tissue is not photosynthetically self-sufficient, and growth regulators; it is solidified with about 0.8% agar for callus and left liquid for suspensions, adjusted to pH 5.6–5.8 and autoclaved. Cultures are kept near 25 °C, usually under a 16-hour photoperiod. A friable callus — one that breaks apart easily — is transferred to liquid medium and shaken (around 100–120 rpm) to give a cell suspension, which is subcultured at regular intervals by diluting part of it into fresh medium.

Growth kinetics. A batch suspension follows a sigmoid curve — lag, exponential, linear, deceleration and stationary phases — and growth is followed by fresh weight, dry weight, packed cell volume, cell number (after separating aggregates) or the fall in medium conductivity as ions are taken up. In the exponential phase the biomass obeys X = X0 e^(μt), so the specific growth rate is μ = ln(X2/X1)/(t2 − t1) and the doubling time is td = ln 2/μ. Plant cells are slow: their doubling times are of the order of a day to several days, against minutes to hours for bacteria — which is why contamination is the commonest reason a plant culture fails. The growth index is (final biomass − initial biomass)/initial biomass.

Worked example. A suspension rises from 2 to 8 g per litre dry weight in 6 days of exponential growth. Then μ = ln 4 / 6 = 1.386 / 6 = 0.231 per day, td = 0.693 / 0.231 = 3.0 days (two doublings in six days, as the factor of four says), and the growth index is (8 − 2)/2 = 3.

Nutrient optimisation. The carbon source (sucrose concentration), the nitrogen supply and its ammonium : nitrate balance, phosphate, and the growth-regulator levels are the usual variables, and as with microbial media they are screened statistically rather than one at a time. Two facts recur in exam questions. First, growth and product formation often need different media: phosphate or nitrogen limitation and high sucrose tend to stop growth and switch on secondary metabolism, so a two-stage culture grows biomass in a growth medium and then transfers it to a production medium. Second, plant cells are large, vacuolated and shear-sensitive, aggregate into clumps and have modest oxygen demand, so they are grown in low-shear vessels — airlift bioreactors or stirred tanks with gentle impellers — rather than the vigorous stirred tanks used for bacteria.

Secondary metabolites are compounds not needed for basic growth but important for defence, signalling and colour, and they are the reason plant cell culture is an industry. The three big classes are alkaloids (nitrogen-containing — vinblastine and vincristine from Catharanthus roseus, morphine, nicotine), terpenoids (from isoprene units — paclitaxel from Taxus, artemisinin from Artemisia annua) and phenolics (from the shikimate and phenylpropanoid route — flavonoids, anthocyanins, the naphthoquinone pigment shikonin). Their synthesis is usually non-growth-associated: it rises as growth slows, is often tissue-specific (some compounds are made only in roots or in specialised glands), and can be low or unstable in undifferentiated cells.

• Select high-yielding cell lines — cultures are heterogeneous, so clones are screened and the best producers kept.
• Optimise the medium and run two stages — a growth medium, then a production medium (Section 3).
• Elicit — add methyl jasmonate, chitosan or a fungal extract late in growth.
• Feed precursors — supply an early intermediate of the pathway (for example phenylalanine for phenylpropanoids) when its supply limits the flux.
• Immobilise and permeabilise — entrapped cells grow slowly and produce longer; permeabilising agents release products stored in the vacuole.
• Remove the product in situ — a second liquid phase or an adsorbent resin takes up the product, relieving feedback inhibition and toxicity.

Hairy root culture uses Agrobacterium rhizogenes (now classified as Rhizobium rhizogenes). Its Ri (root-inducing) plasmid transfers a T-DNA carrying the rol genes (rolA, rolB, rolC) into the plant genome at a wound, and the transformed cells proliferate as highly branched roots. Hairy roots grow fast without added growth regulators, are genetically and biochemically stable over many subcultures (unlike callus and suspension cells), and make the same metabolites as normal roots of the species — tropane alkaloids such as hyoscyamine and scopolamine, ginsenosides, and others whose synthesis needs root differentiation. Their drawback is engineering: a tangled root mass is hard to grow at scale, so mist and bubble-column reactors are used.

Plant products of industrial importance include pharmaceuticals (paclitaxel, now made commercially by plant cell fermentation; vinca alkaloids; artemisinin; codeine and morphine), dyes and pigments (shikonin — the first secondary metabolite made commercially by plant cell culture, in Japan in the early 1980s — and anthocyanins), flavours and sweeteners (vanillin, steviosides), natural insecticides (pyrethrins, azadirachtin from neem), enzymes (papain from papaya latex, bromelain), and industrial raw materials (natural rubber, vegetable oils, starch, fibres). Plant cells are also hosts for recombinant proteins (molecular farming): taliglucerase alfa, a replacement enzyme for Gaucher disease made in carrot cell suspensions, was approved by the US FDA in 2012 as the first plant-cell-made biopharmaceutical.

Artificial (synthetic) seeds are somatic embryos — or shoot buds and other propagules — encapsulated in a protective gel so that they can be handled and sown like seeds. The standard method drops each embryo in sodium alginate (a few per cent) into calcium chloride solution; calcium ions cross-link the alginate into an insoluble calcium alginate bead around it, and nutrients, growth regulators or fungicides can be included as an artificial endosperm. Hydrated synthetic seeds must be sown soon; desiccated ones, made from embryos matured with ABA to tolerate drying, can be stored. They are used for elite hybrids and sterile or seedless genotypes that cannot be propagated through true seed, and for germplasm exchange and conservation.

Somaclonal variation — the term Larkin and Scowcroft introduced in 1981 — is the genetic and epigenetic variation seen among plants regenerated from tissue culture. Its causes are chromosomal changes (aneuploidy, polyploidy, rearrangements), point mutations, activation of transposable elements, gene amplification and changes in DNA methylation. It increases with a long callus phase, many subcultures, strong auxins such as 2,4-D, and indirect rather than direct regeneration; the variation arising from anther or pollen culture is called gametoclonal. It is a nuisance in clonal micropropagation, where plants must be true to type, but a source of useful variants — disease resistance, stress tolerance — for breeders, who screen regenerants for them.

Protoplasts are plant cells whose cell wall has been removed, usually enzymatically with cellulase and pectinase (macerozyme) in an osmoticum (mannitol or sorbitol at roughly 0.4 to 0.6 M) so that the naked cells do not burst; viability is checked with fluorescein diacetate, which only living cells convert to fluorescent fluorescein. Protoplasts take up DNA, and they can be fused: by polyethylene glycol (with high pH and calcium) or by electrofusion (an alternating field lines cells up, a brief pulse fuses them). Fusion bypasses sexual incompatibility. When the nuclei of the two partners fuse as well as their cytoplasms, the regenerated plant is a somatic hybrid carrying both nuclear genomes — its chromosome number is the sum of the parents’ (the potato–tomato “pomato”, 48 + 24 = 72). When only the cytoplasms combine and one partner’s nucleus is lost — deliberately, by irradiating or enucleating it before fusion — the product is a cybrid (cytoplasmic hybrid): one parent’s nucleus with organelles from the other. Cybrids are the way to move cytoplasmically inherited traits such as cytoplasmic male sterility or chloroplast-encoded herbicide resistance into an elite nuclear background.

Indirect gene transfer uses Agrobacterium tumefaciens, which causes crown gall by transferring part of its Ti (tumour-inducing) plasmid into plant cells. The transferred part, the T-DNA, is defined only by its 25-bp left and right border repeats; in the wild it carries genes for auxin and cytokinin synthesis (hence the tumour) and for opines that feed the bacterium. Transfer is driven by the vir genes elsewhere on the plasmid: VirA, a membrane sensor kinase, detects phenolics such as acetosyringone released by wounded cells and activates VirG, which switches on the other vir operons; VirD1 and VirD2 nick the borders and VirD2 stays covalently bound to the 5-prime end of the single-stranded T-strand, which VirE2 coats; the VirB proteins build the type IV secretion channel through which it passes. For engineering the T-DNA is disarmed — the tumour genes are removed and the gene of interest and a selection marker are placed between the borders. In a binary vector system the small T-DNA vector, which replicates in both E. coli and Agrobacterium, is separate from a helper Ti plasmid that supplies the vir genes in trans; the older co-integrate system recombined the construct into a disarmed Ti plasmid. Leaf discs are co-cultivated with the bacterium and then moved to medium with a selective agent and an antibiotic that kills the bacterium; Arabidopsis is transformed simply by dipping flowers (floral dip).

Direct gene transfer delivers naked DNA without a biological vector. Particle bombardment (biolistics) fires gold or tungsten microparticles coated with DNA into intact tissue with a helium-driven gun — the method that first transformed cereals and the only routine route into plastids. Electroporation opens transient pores with an electric pulse, and PEG-mediated uptake works on protoplasts; microinjection, silicon carbide whiskers and liposomes are less common. Direct methods are genotype-independent and need no Agrobacterium host range, but they tend to insert multiple, rearranged copies, which favours gene silencing; Agrobacterium usually gives fewer, cleaner insertions. India’s Bt cotton (a cry gene for an insecticidal Bacillus thuringiensis protein, approved in 2002) and Golden Rice (phytoene synthase and a bacterial carotene desaturase for provitamin-A carotene in the grain) are the transgenic plants an exam is most likely to name.

Marker genes need not stay in a released crop: they can be removed by co-transformation with the marker on a separate T-DNA that segregates away in the next generation, or by site-specific excision with Cre recombinase acting on flanking loxP sites. Plastid (chloroplast) transformation inserts genes into the plastid genome instead of the nucleus. DNA is delivered by biolistics (or PEG into protoplasts) and integrates by homologous recombination between plastid sequences flanking the transgene, so insertion is precise and position effects are absent. Selection uses *aadA* (aminoglycoside adenylyltransferase, spectinomycin and streptomycin resistance), and because a leaf cell holds many plastids, each with many genome copies, several rounds of regeneration on selective medium are needed to reach homoplasmy — every copy transformed. The advantages are very high expression (from the many copies), expression of several genes as one operon, no gene silencing, and gene containment, because plastids are maternally inherited in most crops and so are not spread by pollen. The limitations are that plastids do not glycosylate proteins, and routine plastid transformation is established in only a few species, tobacco above all.