Magnetising Pollen to break the Plant Transformation Bottleneck?
The potential for crop improvement by ‘traditional’ genetic modification and by ‘game-changing’ gene-editing technologies is easy to appreciate. The introduction a foreign gene or the alteration of endogenous gene function in order to modify the way in which a plant responds to a particular environmental stimuli is the underlying goal of most applied plant scientists. Our improved knowledge of how these techniques work, advances in the speed and cost DNA synthesis alongside the adoption of the principles of synthetic biology in the engineering of molecular constructs means that generation of DNA parts for genetic modification is, in theory at least, now facile endeavor.
However there is an ‘eleplant in the room’ of every grant proposal that promises to generate an altered variety of any desired orphan crop. Our ‘eleplant’ is the efficiency, or lack thereof, in plant transformation. This issue was the topic of a 2016 Perspective piece in The Plant Cell in which the example of Sorghum was cited, an important food crop that is unfortunately recalcitrant to transformation, taking up to 12months to generate T1 transformants. This bottleneck will continue to be an issue when discussing new targets for genetic modification as callus-based mechanisms of transformation are famously extremely challenging, with one method good for the goose might be not so good for the gander.
These challenges have been solved for many major crops but even with this knowledge, regeneration of transgenic crops only usually takes place in labs with specific knowledge and experimental pipelines (in the UK at facilities at NIAB or the JIC)*.
It is in this climate that a recent paper by Zhao et al in Nature Plants might be another true game changer. They have modified the magnetofection procedure that has been used very successfully to introduce DNA into animal cells in order to modify existing pollen transformation techniques. This protocol involves mixing DNA with magnetic nanoparticles that can be introduced using a magnetic field into pollen grains through small apertures in the pollen wall. These transformed pollen can then be used to fertilise emasculated plants as normal, from which transgenic seeds can then be selected in the usual manner.
This technique relies upon the pollen aperature being greater than 5um and Zhao et al demonstrate that this was possible in a range of flowering plants including pepper, pumpkin, zucchini and lily. The majority of their experimental work highlighted the introduction of a gene expressing BT toxin into cotton and the subsequent identification of insect resistant plants. The viability of magnetotransformed pollen was unaffected and after the initial fertilization the transgene segregated with normal mendelian ratios.
Importantly for future uptake of this technology, the authors were able to successfully transform Elite varieties that are recalcitrant to callus-transformation, thus greatly reducing the time usually needed for crossing between easily transformable and elite lines. Success rates even for floral dip transformation are lower than 1% so the reported 2-10% in this study, over three years of experiments, strongly suggests that this technique has enormous potential for crop genetic modification.
The only minor drawback is that due to the high success rate, extra generations of selfing transgenic plants might be necessary to obtain pure breeding lines due to the integration of multiple independent insertions.
These experiments have been conducted with a single research lab so it remains to be seen whether these success rates are recapitulated in other locations that have similar but potential significant alterations in their experimental setup.
Importantly the authors do not attempt to use this technique to transform any grass species, a taxonomic group that supplies the vast majority of global animal calories. This will be important to ascertain yet might prove challenging or impossible due to the size of grass pollen grains. Only time will tell whether this is possible.
There is little doubt that this work will raise significant interest in academic and industrial labs across the globe.
Watch this space whether this will prove the breaking of the transformation bottle(neck).
*- Of course Arabidopsis is immune from such concerns as it can be transformed by floral dip, due to an unusually open gynoecium during development.
A commentary article on the Zhao et al paper is also available in Nature Plants.
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