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Micropropagation (tissue culture) · Hydroponics Daily

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Hi, welcome back to Hydroponics Daily with me, Dr. Russell Sharp.
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Yesterday's topic was polyploidy and I said that today we'll be talking about
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micropropagation and we will.
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I just want to revisit polyploidy for a second because I missed a couple of key bits out.
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The first one is, how do you confirm your plant is polyploidy?
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Maybe it's just a mutant.
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And what you have to do is you take a microscope slide
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of some of the cells of your plant and you
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stain them with a chemical called saffronin and this
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is a chemical that's extracted from saffron as you would imagine because
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of the name it's the red dye in saffron however you
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can just buy the pigment you don't need to extract it and then you just stain
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the cells and then the chromosomes the dna takes it up and then you can count
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them really easily in the nucleus of the cells and that will confirm whether
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they have artificially increased the chromosome count or if they're bet back
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at what the wild type plant should be.
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You might also want to do this to confirm the chromosome numbers so if you're
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looking to cross them and things like this however that's fairly advanced and
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so is taking microscope slides it's not something you just whack under a microscope
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and and do fairly easily so you need to be pretty good at using a microscope
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to to use those sorts of techniques.
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This technique that I used this was my first job ever in plant science was counting
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chromosome numbers on microscope slides and that was at Ness Botanical Gardens
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on the Wirral Peninsula.
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It's part of Liverpool University and my tutor there was Professor Hugh McAllister.
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Shout out to Hugh McAllister and all the other plant scientists and hydroponic growers in Liverpool.
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Liverpool is a hotbed of hydroponics so I'm sure we've got some listeners on Merseyside.
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I'm from Merseyside originally but unlike Stephen Barry from NPK Hydroponics
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podcast I don't have a Scouse accent and that's because I went to university
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to study plant scientist and nobody could understand my accent so I had to change my accent.
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All my family speak with Scouse accents especially my sister but yeah I'm a
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dad but I don't speak with a Scouse accent but I am from Liverpool originally and.
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Yeah, so that's polyploidy. We'll move on to micropropagation now.
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It's a technique linked to polyploidy because you can induce polyploidy very
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easily if you apply the chemical colchicine to the calluses,
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the undifferentiated growth, the blobs of growth that you get in micropropagation.
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However, that's not the only reason you would want to micropropagate a plant.
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There are loads of reasons you might want to micropropagate a plant.
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And that's normally because either you're struggling to propagate them from
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cuttings or grafting or you're having little success in germinating seeds or
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it takes a long time using conventional methods.
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These are all reasons you might want to micropropagate a plant.
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Another term for micropropagation is tissue culture. So if you're struggling
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to find resources on micropropagation you'll also find stuff on tissue cultures or microprop for short.
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Okay so in its simplest form it's producing new
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plants from small amounts of tissues excised from
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the mother plant under sterile conditions and
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cultured on a gel medium and by far
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the most important word there is sterile because
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it's a bit like brewing in micropropagation you
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spend 99% of your time cleaning things
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and making sure that everything is sterile same in brewing brewing
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is just cleaning stuff and it's
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it's a really good technique it's very
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scientific and there are there are
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a few amateur micropropagation kits now
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available athena i started selling they're
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quite expensive i think they were about a thousand pounds and everyone
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there's a big hype around them not sure if anyone used them successfully to micropropagate
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any plants when they were released a couple of years ago and you might think
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oh that athena they're making loads of money out of that but it's a fairly complicated
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kit the ingredients the materials are not cheap and getting them all together
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couldn't have been easy so you know fair play to athena for putting it together
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no one else was making micro propagation.
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Kits and i would imagine that was probably a lost leader
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just to make sure that they were seen as the sort
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of leaders in scientific growth in hydroponics
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so you know you know kudos to them for trying you
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a competitor but at least they did something different and then
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so micropropagation so you may have heard of micropropagation in
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your industry it might be some niche users or it might be standard i mean if
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you're cultivating orchids carnivorous plants ferns and palms bamboos then micropropagation
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is the standard way they are propagated there is a company called Micropropagation
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Services in Nottinghamshire,
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and they micropropagate sphagnum moss,
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and they then put them into little beads of agar,
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and they get into a helicopter, and they sort of fire them out of a helicopter
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like it's Vietnam in the 1960s and 70s, and they pepper these moss balls all over...
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Peat bogs and moorland to reintroduce sphagnum moss, which is a key structural
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species in these environments.
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Really interesting one. Another example of where it's been used to propagate
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rare plants is the yellow lady slipper orchid, which in the 1990s,
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I think it went down to about one plant in the wild.
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And then Kew Gardens, the biggest botanical gardens in the world,
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used mangro propagation to raise the plants of this rare orchid.
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But it's used on a lot of asian orchid nurseries
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and things like that it's the standard way see i've seen micropropagation used
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on bamboo and orchid nurseries in holland and many
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times and this is an amazing fact if
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you have a micropropagation lab of
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23 square meters which is
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not big that is a standard bedroom you can
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produce up to a million plants annually so
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that is crazy numbers and with
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an initial outlay you can do this for under two thousand pounds or
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three thousand dollars but you know these
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setups can run up to hundreds of thousands even millions of
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pounds when you start putting in the the
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grow on and the greenhouses and the grow rooms to actually
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get those into bigger plants one of the words you'll hear
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often in micropropagation is weaning and just
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like a baby weaning them off milk you have to wean them
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off the agar and the the sterile environment that's
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what the specialist greenhouses are for is that
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you need a you need to wean them slowly in very high humidity and then lower
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the humidity as the stomata become adapted to this and the growth forms you
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can't just keep them forever in 100 humidity another before i go into the details
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of how you actually micropropagate there's some other good uses for micropropagation
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is the eradication of viruses from infected stock.
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So imagine you've got a strain, a variety that is really popular in your industry,
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but the mother plants get infected with a disease.
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And then you can use micropropagation to remove the virus. It's a very good
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way of removing viruses.
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And so things like hops, things that get riddled with viruses,
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that would be a good option.
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So we said weaning is a word that's used often. another word that's used in
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micropropagation is one of my favourite words in science, and that is totipotent.
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Totipotent means that basically a meristematic tissue, a plant from the growing bud,
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the meristem of the plant, can turn into any
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of the cells programmed in the plant's genome so
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it's so you could that's that that undifferentiated cell could turn into it
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was from a carrot plant it could turn into a carrot root it could turn into
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a root hair it could turn into a xylem vessel it could turn into a phloem vessel it could turn into.
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A leaf stomata sorry not
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a stomata stomata just the whole a guard cell could turn
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into a petal it could turn into a pollen tube it
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could turn into anything and that was discovered by a
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british botanist frederick stewart working at
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cornell university in new york in the 1950s and
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he showed that you could produce a complete carrot plant from just
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a single cell so that is 40 years
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before the first animal was ever cloned
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that was dolly the sheep produced from a single mammary
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gland cell up in scotland so plant scientists
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have been doing this for years before the human scientists
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get involved same with virology you know plant
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science has been talking about their virus studies are way in advance of human
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viruses and animal virology for a long time so it doesn't matter where the cells
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come from in the plants unlike animal cells where there's sort of like a fixed
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destiny on the cells having said that meristem cells are really the ones you
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want to produce new plants from.
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So when you're taking plants for micropropagation, when you're using a sterile
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scalpel, the first thing you do is you remove the meristematic tissue from the
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flower buds, probably an active growing one.
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You remove the leaves, the primordial, these are the young leaves around the
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stem, and then you need a microscope to dissect it, and then you put it onto.
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Gel on a petri dish or in a petri in
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a tube with a agar gel or some other
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sort of gel nutrient stock in there and then
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just lay it on top and keep it in that and
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seal it keep it all sterile and everything has to be sterile the
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media has to be sterile and so you use techniques like
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using ipa isopropyl alcohol you
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have a autoclave which is basically like a
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pressure cooker where you make sure
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everything's sterile by heating it up under pressure and the
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first grow media is normally just a standard grow media
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with some nutrients in and some food for
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the what they call the x plants the these little
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blobs that you've taken from from the merostem and you
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need a bit of sugars in there because they're not
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going to be photosynthesizing at all so they're going to be growing just like
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an animal they're going to be feeding like you know
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like a fungus or something like that and they're just
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going to be multiplying by mitosis and they're
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not going to be differentiating and then eventually the clump
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will grow and grow and grow and it'll form a callus just like a callus on your
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foot or your hand differentiated a bit like a tumor you know it's not differentiated
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undifferentiated and you just want to build that callus up you don't want it
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to turn into a plant too soon because it needs to have energy reserves so that
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when it does produce roots and shoots,
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that it's got the energy to do so.
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And maintain it. So as I say, the media contains all sorts of things like amino
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acids as well to make proteins.
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The most famous one is called Murashigi and Skoog, and that's named after the
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famous plant scientists Toshio Murashigi.
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Murashigi and Folkashkug. They are the Lennon and McCartney of micropropagation.
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Together, Murashigi and Skoog published the earliest definition of the technique
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in the early 1960s and developed grow media to culture the tissue on and that
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grow media is still used to this day and the recipes have been tweaked but they're
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largely unaltered and a lot of people still use muragashian skug as known as
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mso in my propagation labs all over the world to this day.
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So the first one that you put the the x plant on be high in auxins and just like auxins in,
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in plants and rooting and and
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rooting hormone auxin is it will
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will stimulate rooting before you
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stimulate rooting though you might want to cut
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the callus up again especially if you haven't got much of
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the starting material to play with so you know
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that example of the the yellow slipper orchid
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you obviously can't be coming back and back to the
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one plant to rip it up to take cultures so once
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you've got a bit of a big callus you might
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then use your sterile scalpel to subculture and
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make more calluses and then build them up and keep those calluses
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going but then you say you have
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stimulated the the roots to form with with the
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hormones the auxin then it's time to
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transfer them to a new gel medium with cytokinin hormones
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and they stimulate the production of shoot tissue and then after that you've
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got everything you need for a plant you don't need to stimulate flowers and
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fruits obviously in a in a test tube or a petri dish and interesting story about
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petri dishes i used to work at a place called malton college part of the northampton university,
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and there was a student there who was called petri.
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And oh petri and i said oh
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you named petri after petri dish and she
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got really upset and mum phoned in and said this is disgraceful
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how the teacher was bullying her and i
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said that's absolute nonsense you should have seen it as a compliment named
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after a famous scientist petri who invented the
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petri dish and was a famous microbiologist and yeah
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going a bit of trouble for that one but yeah you know
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f-r in the a to be honest you know you're gonna
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be called petri you've got to be expect to be called petri dish every now and
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again where do you store all these plants while
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you're all these calluses these petri dishes and large
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test tubes they're not really a test tube they're more like a boiling tube with cotton
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in the top that allows the air to flow out and in but prevents
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dust and bits of bacteria from getting in you
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probably want to put them in a controlled environment it's a
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bit like a grow room but much more cleaner no growing
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media around no stagnant water so maybe
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like a and then you can get um like almost
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like fridges with a little grow light in there they don't
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tend to use leds or sodiums certainly not it's more
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fluorescent lights in micropropagation is fairly standard and you just put them
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in there until they they grow to the point where they start to outgrow the boiling
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tube or the test tube photosynthesis will be very low because the plants are
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in 100 humidity they haven't got much water.
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Haven't got and and so there isn't much co2 really
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as well so growth is very very limited and
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slow in micropropagation so it can take up to two to four months to form a plant
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in micropropagation but you can clean the good thing about micropropagation
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is you can keep them in sort of stasis as well and so you could use them as
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a sort of safety net for your wonder plants and as a sort of.
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I want to say repository. I always get confused between repository and suppository.
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As a repository for all your different strains and varieties, things like this.
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Whereas if you were storing them as fully grown plants, that would obviously take up a lot of room.
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So imagine like a seed bank, but for living plants.
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So you use the autoclave to sterilize all the kit that's going to be in close
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contact with the plants.
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You would also sterilize all
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the the cotton wool that you're putting into the the boiling tubes the gray
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media would be autoclaved as well but then the things like the hormones you
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don't want to autoclaved them because they would break down the amino acids
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would break down maybe under high temperatures so you could you could filter
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sterilize them which is another,
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pain and another cost and but is
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required the actual explant you would
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sterilize in either ethanol or dilute bleach or something
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like this probably ethanol but that is going to
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be the source of most contamination because that's where you know it's going
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to be much harder to sterilize and if the plant has got a disease or it's got
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bacteria living in it endophytes they're they might not be harmful but they
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might be living there then they can be a source of infection so So that's really your,
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this can be the source where most infections are going to come from in micropropagation.
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Often you use something called a laminar flow hood.
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It's almost like the opposite of a fume cupboard if you've been to a laboratory. This one pushes...
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Sterile air through at you as you sat next
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to the cupboard and it's all filtered and
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so all this air comes at you rather than being pulled up away from you
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like you would in a normal fume cupboard to take hazardous fumes in
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a chemical laboratory but there's all
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this air flowing towards you you've got ethanol and you might have
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a flame to sterilize your scalpel and
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stuff like that so it's a bit of a fire hazard once i
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was working with a colleague called yannick down at east moorling in
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kent and he spilled his ethanol and it all went on fire
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and he looked at him next to him and said oh adam i've
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got a problem it's like yeah you have a fire extinguisher
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and he was on fire in this micro propagation lab where
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they were growing trees for growing it for shell something
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like that for one of their alternatives to petrol petrochemicals poplars
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they were growing that was it yeah so it can also be used
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to increase the sort of active ingredient
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levels of plants there's interest in sort of like from
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the pharmaceutical industry using tissue culture to synthesize
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important drugs so examples like
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the madagascan periwinkle that's used for leukemia
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and hodgkin's disease but you know
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currently it requires tons of plant tissue to obtain just
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two grams of active ingredients so you could massively increase the
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active ingredient content that would be useful and
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certainly other plants where you've got medical use
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then they could potentially be used for micropropagation therefore
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not just for propagation but for also for increasing content.
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Of the plants it's important to know
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just like cuttings you always want to take the x
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plants from juvenile plants not mature plants
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that are flowered because it's always going to be harder to propagate
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from plants that have flowered as soon as they as soon
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as they've become mature they're a lot harder to propagate asexually it's just
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something goes on in the plant not quite sure what but then routinely it's going
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to be an issue and as as with all sorts of forms of asexual propagation the
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plants produced by micropropagation are clones of the mother plant.
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However mutations are common when they're in
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the callous stage so and polyploidy
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as we talked about in the previous episode and the start of this one can occur
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in polyploidy and especially if you stress them if you put chemicals on
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like colchicine uv light x-rays gamma
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rays things like this can all be used to treat plants in
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callous stages and you can get mutations most
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and 99% of all mutations are deleterious however 1% at the time you can get
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a nice good one in plants and it's not like mutating humans it's there's no
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ethical issues because they're not going to feel pain or you know have issues
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long term it's just a plant,
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One notable success of micropropagation has been the rare Tunbridge filmy fern,
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which was thought to be close to extinction in the town of Tunbridge Wells in Kent,
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and was extinct in Tunbridge Wells until 1875, and then micropropagation has
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been able to increase the numbers dramatically.
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Ferns are difficult to propagate because they don't produce seeds and flowers,
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they produce spores and they have a haploid phase of
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their life cycle where they grow as a haploid for a bit and so
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a single set of their genes so really difficult to propagate ferns unless
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it's sort of asexually so the tunbridge
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filmy fern was a big success story oh yeah
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i didn't mention the when a mutation or a
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growth form that's new not often it's often not
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a genetic mutation just a growth condition like a
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growth deformity occurs in plants it's cut in
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micropropagation it's called somaclonal variation and they
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pop up every now and again in micropropagation especially in the large micropropagation
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laboratories and they have
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been the main source of new varieties of hostas
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and heucheras in gardens over the last 30
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years heucheras especially hostas have
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always been popular gardens especially in the uk where it's so
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wet and hostas really thrive but
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hookahs are fairly i'd say
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in the last 20 30 years really really popular and
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so the new varieties are often coming from the asian
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micro propagators of these plants and all the different colors and
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growth forms so lots like almost like a rainbow of.
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Different hookahs now because of this three more things to
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consider i've been a bit and back and forward in and out
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on this episode it's not really gone in a linear form so
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please bear with me if you want to eliminate viruses using
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micropropagation which is probably what most listeners will want
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to do i don't think many people are looking to breed
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or mutate if you do want to do virus treatment you
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want to heat you might want to flash heat that that
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callus and to maybe 50 60 degrees celsius
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and that should that could almost destroy
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the virus but not kill the plant that's what you're looking for with
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virus treatments and same with seeds the seedlings
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often if you flash heat them really quickly
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then that can kill the virus and not kill the plant when it
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comes to the weaning you might want to include a mycorrhizal inoculants because
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remember they're going from a sterile environment back to an unsterile one and
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they you can't just rely on them to grow media having the the optimum fungus
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in there so that'd be a good one and then even if your plant does propagate from cuttings.
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If it's really, really slow, then micropropagation could be an option as well.
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So the houseplants, the large leaf figs, they can be better micropropagated
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because they just take so long to propagate normally.
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Other plants that would struggle or take a long time, like any sort of woody
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shrub like rhododendron or something like that, can take ages,
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even if you're really good at propagating them.
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So that's been Hydroponics Daily for today. micro propagation
22:00
or tissue culture it's also routinely known
22:03
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sort you out. Thank you very much for listening.
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I've been Russell Sharp. This has been Hydroponics Daily, and you've been an
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amazing hydroponic grower. Thank you very much. See you tomorrow.