Protoplast fusion and transgenic plants

A protoplast is a plant cell whose cell wall has been removed, leaving the plasma membrane as the outer boundary. With no wall it can take up DNA, fuse with another protoplast and, in suitable culture, rebuild a wall and divide. Protoplasts are totipotent in principle: a cultured protoplast can form a callus and then a whole plant, although how readily plants regenerate depends strongly on the species and genotype.

Protoplasts of two parents are brought together and induced to fuse. This allows crosses between species that cannot be crossed sexually, because it bypasses the pre-fertilisation barriers. Incompatibility after fusion can still stop the product from growing. Fusing protoplasts of different parents gives a heterokaryon: the nuclei of both parents in a shared cytoplasm. Fusing two protoplasts of the same parent gives a homokaryon. If the nuclei of a heterokaryon fuse (a synkaryon), the cell becomes a somatic hybrid. If one nucleus is lost or inactivated, the product can be a cybrid.

Selection of fusion products. Unfused parental protoplasts and homokaryons must be removed. Common strategies are: (1) complementation, where each parent is blocked in a different way so only the hybrid grows. For example, one parent is an albino mutant that can regenerate, and the other is green but cannot regenerate in the medium used; or the two parents carry different non-allelic mutations. (2) Metabolic inactivation plus irradiation. The parent that will supply the nucleus is treated with a metabolic inhibitor, such as iodoacetate or iodoacetamide (or rhodamine 6G, which inactivates mitochondria). The other parent is given X-ray or gamma irradiation to inactivate its nucleus. Neither parent can divide alone, but the fusion product can. The irradiated parent supplies cytoplasm (cybrid) or a few chromosome fragments (asymmetric hybrid). (3) Fluorescent labelling of the two parents with different dyes, such as fluorescein isothiocyanate (green) and rhodamine isothiocyanate (red), or the natural red chlorophyll fluorescence of mesophyll protoplasts. The dual-labelled fusion products are then sorted with a fluorescence-activated cell sorter or picked with a micromanipulator. (4) Differences in morphology or resistance, with later confirmation by isozyme patterns, chromosome counts or molecular markers.

A well-known example is the pomato, a somatic hybrid of potato and tomato, which is of scientific interest but not a useful crop. Cybrids have been used to transfer cytoplasmic male sterility between related plants, since this trait is carried by mitochondrial genes. In Brassica, cybrids have also been used to transfer triazine (atrazine) herbicide resistance, which is encoded in the chloroplast genome.

Agrobacterium tumefaciens is a soil bacterium that causes crown gall disease. It carries a large Ti (tumour-inducing) plasmid. A segment of this plasmid, the T-DNA, is transferred into the plant cell and integrates into the nuclear genome. There its genes make the plant produce the auxin indole-3-acetic acid (IAA; genes iaaM, tryptophan monooxygenase, and iaaH, indoleacetamide hydrolase), a cytokinin (gene ipt, isopentenyl transferase) and opines, which the bacterium uses as food. The vir genes that carry out the transfer lie on the Ti plasmid outside the T-DNA, and chromosomal genes of the bacterium (chv genes) are needed for attachment to the plant cell. In genetic engineering the tumour-causing T-DNA genes are replaced with the gene of interest, and the strain is said to be disarmed. Only the two border sequences of the T-DNA are needed in cis for transfer, not the genes between them; the vir genes act in trans.

Agrobacterium infects dicots naturally. Monocots, such as cereals, are less susceptible, partly because their wounded tissues produce little of the phenolic inducers. They can still be transformed by adding acetosyringone, using highly virulent strains or super-binary vectors (which carry extra vir genes), and choosing suitable explants such as immature embryos or callus. For Arabidopsis, the floral dip method transforms developing ovules in the intact plant, so tissue culture is not needed.

Agrobacterium rhizogenes carries the Ri (root-inducing) plasmid. Its T-DNA carries rol genes, which cause hairy root disease. Hairy root cultures grow fast without added growth regulators and are used to produce secondary metabolites. The Ri plasmid can also serve as a vector.

Compared with Agrobacterium, direct methods have no host-range limit and need no bacterial vector. However, the DNA often integrates as multiple, rearranged copies, which makes silencing and unstable inheritance more likely. Agrobacterium tends to give fewer copies with defined borders.

Only a small fraction of treated cells take up and integrate the transgene. A selectable marker gene allows only the transformed cells to survive or grow under a selective agent. A reporter gene encodes an easily assayed product. It is used to check where and how strongly a gene or promoter is expressed, or to identify transformed tissue without killing untransformed cells.

After selection, transgenic plants are confirmed by PCR (presence of the transgene), Southern hybridisation (integration into the genome and copy number) and checks of expression (RT-PCR, northern or western blots, or enzyme assays). Where a plant should not keep its marker, the marker can be removed with the Cre-lox system. It can also be avoided by co-transformation: the marker and the gene of interest go on separate T-DNAs and later segregate apart.

The plastid genome (plastome) of flowering plants is a circular DNA of roughly 120 to 160 kb. A leaf cell has many chloroplasts and each has many copies of the plastome, so one cell can hold thousands of copies. In plastid transformation the transgene, flanked by plastome sequences, is delivered by particle bombardment (or by PEG treatment of protoplasts) and integrates by homologous recombination at a defined site. A plastid marker such as aadA (spectinomycin resistance) is used. Repeated rounds of selection on regeneration medium are needed to reach homoplasmy, where every plastome copy carries the transgene.

Examples of transgenic crops and traits at GATE level: Bt cotton and Bt maize (the cry genes of Bacillus thuringiensis give insect resistance) and herbicide-tolerant crops (a glyphosate-insensitive EPSPS or the bar gene). Golden Rice carries a plant phytoene synthase (psy) and a bacterial carotene desaturase (crtI), which give beta-carotene in the grain. Virus-resistant papaya carries a viral coat protein gene. In the Flavr Savr tomato, an antisense polygalacturonase gene reduces pectin breakdown and slows softening.