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Micropropagation: A Primer

Growing Plants From Seed

There are several different ways of growing crops, the oldest and simplest being growing plants from seed.  This is still by far the most common method being used today, cereal grains, pulse crops, and vegetables all come from seeds either seeded directly into the soil or started in a nursery and then transplanted.  Large numbers of plants can be produced very quickly when growing from seed. The disadvantage to this method is that since it's a sexual form of reproduction there is no absolute guarantee in genetic purity.  Suppose that there's a true breeding strain being propagated through seed, all it would take is a few grains of pollen to contaminate it as this would show up in the subsequent generations.  Natural selection also plays a factor, if the contaminant offers an advantage then the frequency of these genes will increase over time. 

 

 

Cloning

As the value of the plant goes up, so too does the sophistication and amount of work of the propagation method.  The next method is cloning in which a plant with desired genetics is found and shoots are removed.  These will become the next generation.  The cuttings can be dipped in a rooting hormone and then stuck into a rooting medium, such as soil, perlite, or rockwool.  Usually they're kept in an environment which is high in humidity to keep the plant from drying out since the cutting has no roots to supply water with.  This can be risky since pathogens also like high humidity.  After a few weeks, once the plant has developed roots it can be transplanted to complete its life cycle.  The advantage over seed is that genetic purity can be maintained by cloning the subsequent generations.  In plants which produce both male and female sexes it is possible to maintain stocks of the desired type.  Hybrids can also be used in the successive generations, whereas seed will lose potency after the first generation.  The disadvantage is that mother plants take up a lot of space and can only produce so many clones at one time.

Some plants clone themselves, strawberries and spider plants both send out runners which are shoots that will eventually root themselves and start a new plant.

 

 

Out of the Fields and Nurseries and into the Lab

Micropropagation, (a form of plant tissue culture) has the advantage of seed in that large numbers of plants can be produced in a relatively short amount of time and the same advantage of cloning in that everything will be genetically identical.  The other advantage it has over cloning is that very large numbers of plants can be started in a small amount of space.  The disadvantage of this method is that it takes a higher level of skill, most people can plant seeds or can take a cutting after being given simple instructions.  Micropropagation has multiple steps and different plants have different requirements so way more training is needed as well as specialized equipment.  The other factor that can complicate things is the fact that everything needs to be completely sterile as mould can easily overgrow plantlets. 

 

*Did you know?*

There are several types of plant tissue culture, one of the more interesting ones is called microspore culture.  Immature pollen cells are extracted from developing flower buds and are grown up in culture.  Normal cells have 2 sets of chromosomes (diploid) and microspores will only have one (haploid), but if the growing plant is treated with a chemical that prevents cell division it could wind up with the proper set of 2.  This has a huge advantage in plant breeding since it's possible to have a true breeding line in only one generation.  Using traditional techniques of inbreeding takes many generations to achieve a true breeding line. 

 

The Process

There are multiple steps in producing plants from micropropagation and this post will go through them quickly, as this post not meant to be a how to guide but rather as a brief introduction to see if the technique is a good option for the readers situation.  The good news is that once one of the steps is well understood the rest are fairly easy since everything is connected.




Media

Just like regular plants, those grown in culture require 12 essential nutrients, but because they're so small and delicate other ingredients are needed as well.  The first being a carbon source, plants will normally take it from the atmosphere but because in culture they're housed in sealed vessels that isn't possible.  Carbon has be given artificially in the form of sugar mixed into the medium.  The other essential ingredients are plant hormones, since they're too small and lack specialized tissues so can't produce their own in any significant amount. 

There are 2 broad categories of media, liquid and solid.  Liquid media is used early on when single cells are dividing to become embryos.  From then on they're grown on a solid media, which is simply a liquid that contains a gelling agent such as agar.

Parent Material to Embryos

The first thing that we need to do is identify a plant with the genetic characteristics of interest and pull off a piece of tissue such as a leaf.  This is put into a chemical disinfectant to sterilize it such as dilute bleach or hydrogen peroxide, keeping in mind that too long and concentrated will kill the plant cells while too short of a time and too diluted won't kill off any mould on the surface of the plant tissue.  

Once sterilized the plant tissue is then macerated usually by crushing it and is then filtered to remove any large pieces of debris left over.  The cells are added to liquid media which is constantly stirred in order to oxygenate the developing embryos.  There are variations on this depending on the species of plant, some require an additional step on solid media before going into the liquid.  Different hormones ratios might also be necessary for different plants as well.

 



Shoots From Embryos

When first extracted the cells will be microscopic in size. They grow into embryos and then get bigger and bigger, eventually becoming large enough to see and handle individually.  At this point they're placed on petri plates of solid media, several to a plate and allowed to continue growing.  Typically medium at this point is higher in cytokinins as these hormones are responsible for triggering the embryo to produce shoots.  

Plantlets...

Growing in cramped petri plates and having less than ideal hormone ratios will produce plants which are deformed.  While there might be shoots and roots there's also a lot of tissue known as callus which are cells that are unspecialized and resemble tumors. 

  

 
Callus material (From Carrot)

 

Normal looking shoots are then cut from the deformed mass and put into new containers.  While these still contain solid medium the containers are tall which allows the plant to grow up and branch out as a normal plant would.  In addition the plants typically also grow a proper root system in this stage of growth.

The Transition to Normal Growth

Once the plantlet has outgrown its container it's time to move it on to the normal part of its life.  The plantlets are taken out of their artificial environment and put into the soil, but at this stage are very delicate and need to be acclimatized.  This is nothing more than some type of humidity dome.  After a few days the dome is lifted a bit so that the plant is exposed to more air and eventually removed all together and then it can be treated like any other plant.

 

 

 

In Conclusion...

As mentioned before, this method of plant propagation is labour intensive and requires specialized equipment and training.  There is a lag time in the beginning as it can be months between the initial cell extraction and having normal plants to transplant.  


On the other hand it should be obvious that using this method it would be relatively easy to have thousands of plants in the pipeline at any one time.  Additionally because everything is sterile there's no concern about contamination from disease and insects.  

 

*Did you know?*

Plant viruses are easily transmitted through cloning and there have been cases where an entire genetic line was contaminated with a virus.  For reasons not entirely clear, the shoot and root tips (apical meristems) don't contain viruses.  If a whole line becomes infected with a virus it is possible to remove the shoot tip and grow it up in culture thus removing the virus from subsequent generations.

 

Mosaic Virus Infection on Potato

 

    Published by Loren Price

    Bio:
    Loren grew up on a mixed grain and cattle farm in north west Saskatchewan. He went on to study biotechnology and worked in the agrosciences in Saskatoon for several years before moving on to Future Harvest and the hydroponic plant food industry. Starting off in fertilizer production his focus is now on fertilizer formulations and regulatory affairs. His areas of expertise include: agronomy, analytical chemistry, plant tissue culture, plant breeding, molecular biology, and plant nutrition. Outside of work Loren collects vintage concert T-shirts and is an amateur craft brewer specializing in historical and lesser known styles of beer.

    E-Mail: loren@futureharvest.com

  • Nov 20, 2018
  • Category: Articles
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