Introducing Protoplasts Part I - The Tortoise and the Hare

by Steven Burgess

‘There once was a speedy hare who bragged about how fast he could run. Tired of hearing him boast, Slow and Steady, the tortoise, challenged him to a race. All the animals in the forest gathered to watch.

Hare ran down the road for a while and then and paused to rest. He looked back at Slow and Steady and cried out, "How do you expect to win this race when you are walking along at your slow, slow pace?"  - Aesop’s fables

Working in plant science I sometimes feel like the tortoise in Aesop's fable. Animal biologists race on ahead making discoveries, before stopping to laugh, amazed plant science's ‘slow, slow pace’. Speed is a real issue when you work with plants, to illustrate the point a colleague of mine recently obtained some seeds that came with the following advice - ‘germination may take between two weeks and two years’. The challenge of time is one of the key reasons why a lot of research is still done in model plant Arabidopsis thaliana: it has a relatively short life cycle, so it can be quite quick to make a mutation and analyse the effect. However, even at 8 weeks to seed, as Arabiodpsis is diploid, so you need to do through the process of growing plants, collecting seed and select mutants three times in order to generate plants suitable for analysis – as a result it is months before you can carry out an experiment.

To speed things up, plant scientists can use transient assays, whereby it is possible to switch on or off a gene of interest for long enough to analyse the effect. The most common methods in plants include agroinfiltration (video), biolistic bombardment (video) or viral induced gene silencing (VIGS). Agroinfiltration uses agrobacterium to infect plants, this gram negative bacterium is able to transfer DNA from itself into a plant genome in a process that has been adapted to introduce a genes of interest. This process works great in Nicotiana benthamiana, but there are many species in which it is far from optimal, and we have not much success using either grasses such as maize, or our model species Gynandropsis gynandra. 

Biolistic bombardment involves coating metal particles with DNA, which are then introduced into plant cells using a genegun (sounds cool, but believe me it gets tedious after a while!). This works okay, and for my purpose it is probably the best option currently available, but biolistics is not really suited to high throughput analyses. VIGS can only be used for switching off genes. 

The alternative is to take an approach that is analogous to using cell lines, the use of which gives animal research its speed. 

Maize mesophyll protoplast

Techniques have been established to create plant cell tissue cultures as well as isolated plant cells known as protoplasts (which do not propagate) from mature leaves, or plant embryonic tissue (known as callus) (if you are interested in the history of protoplasts a personal account is provided by Prof. Edward Cocking who pioneered their use here). However, these approaches have their limitations, the most significant question is how representative of ‘real’ systems are these cells? Plant cell tissue cultures are suitable for biotechnological purposes, but as the gene activation patterns are so far removed from ‘normal’ systems they are not always suitable for scientific study. Protoplasting can introduce stress responses that must be considered when interpreting experiments, but protoplasts are a close enough to real cells to be a useful tool in preliminary analyses. 

Therefore, as previously mentioned, we hope to couple the use of protoplasts to developments in microfluidics to generate a system suitable for high throughput analyses and help give the Slow and Steady plant scientists their running shoes. In the next post we will be give a bit more information about protoplasts and provide an update on our initial attempts to isolate them, as well as preliminary tests about how they behave in a microfluidic system.