Plant Breeding and Genetic Transformation

Plant transformation selection markers

Selectable marker genes allow transformed cells to grow on a medium containing a selective agent, while untransformed cells are killed or stopped from growing. Each marker encodes an enzyme that either detoxifies one specific agent or lets the cell use an alternative metabolite, so the marker and the agent must be matched correctly. Markers are commonly grouped into antibiotic resistance, herbicide tolerance, and positive selection systems.

• nptII — encodes neomycin phosphotransferase II, which inactivates aminoglycosides such as kanamycin and neomycin. Example: a kanamycin-containing medium selects for nptII-carrying calli.
• hpt — encodes hygromycin phosphotransferase, which inactivates the antibiotic hygromycin. It is an antibiotic marker, not a herbicide marker.
• bar / pat — encode phosphinothricin acetyltransferase, which acetylates and detoxifies phosphinothricin (glufosinate), the active part of bialaphos. Because the selective agent is a herbicide, bar confers herbicide tolerance.
• pmi — encodes phosphomannose isomerase, which converts mannose-6-phosphate to fructose-6-phosphate. Transformed cells can therefore use mannose as a carbon source, which is a positive selection system.
• ptxD — allows cells to use phosphite as the sole phosphorus source, another positive selection system unrelated to antibiotics or herbicides.
• dhfr — a dihydrofolate reductase variant insensitive to methotrexate, so the cell keeps folate metabolism running in the presence of the drug.
• EPSPS (glyphosate-tolerant form) — a version of 5-enolpyruvylshikimate-3-phosphate synthase that is not inhibited by glyphosate, so the shikimate pathway continues in the presence of the herbicide.

A common exam trap is to confuse the agent class: hygromycin and kanamycin are antibiotics, whereas glufosinate and glyphosate are herbicides.

Agrobacterium Ti plasmid vir genes

The tumour-inducing (Ti) plasmid of Agrobacterium tumefaciens carries the T-DNA, which is transferred into the plant genome, and a separate virulence (vir) region whose genes supply the proteins needed for transfer. The T-DNA is bounded by 25-bp border repeats, and only these border sequences and the vir gene products are required in trans for DNA processing and export.

The vir genes act in a defined sequence. VirA is a membrane-bound sensor histidine kinase that is expressed constitutively and detects phenolic compounds such as acetosyringone released from wounded plant cells. After autophosphorylation, VirA transfers the phosphate to VirG, a response regulator that acts as a transcriptional activator of the other vir operons.

VirD2 is an endonuclease that nicks the T-DNA border sequences, while VirD1 is a topoisomerase-like protein that assists in this nicking. The processed single-stranded T-strand is then coated by VirE2, a single-stranded DNA-binding protein that protects it during transfer and helps it enter the plant nucleus. The VirB operons encode membrane proteins that form a type IV secretion channel through which the T-complex passes into the plant cell.

A useful check is to ask what each protein does in the transfer cascade: sensing, regulating, cutting, coating, or exporting the DNA.

Golden rice carotenoid biosynthesis

Carotenoids are isoprenoid pigments synthesised from geranylgeranyl diphosphate (GGPP). Rice endosperm normally lacks the enzymes needed to make \beta-carotene, the provitamin A precursor, so Golden Rice was engineered by introducing the missing steps into the endosperm. The core pathway runs from GGPP to phytoene, then to lycopene, and finally to \beta-carotene through cyclisation.

Phytoene synthase catalyses the first committed step by condensing two GGPP molecules to form phytoene. Overexpressing it therefore increases the carbon flux into the carotenoid branch. Carotene desaturase introduces double bonds into phytoene to produce lycopene, the immediate precursor of \beta-carotene, so raising its activity also increases pathway flux toward \beta-carotene.

Enzymes that do not act in carotenoid synthesis do not change \beta-carotene content, even if they are present in a transgenic construct. Reporter enzymes such as \beta-glucuronidase only mark gene expression, and enzymes of the shikimate pathway such as EPSPS affect aromatic amino acid synthesis rather than pigment production.