Meet the Museum: Conserving Plants with Science

Arlene Moncrieff: Hi everyone, welcome to make the Museum on this Thursday night. Cool night it will be tonight. Very much appreciated for you to turn up and see this, what we're really looking forward to in this Meet the Museum presentation with Bryn Funnekotter from Kings Park called Conserving Plants with Science. I've got a few words here about Bryn, which I'll read out to you. Bryn is a research scientist at the Department of Biodiversity Conservation and Attractions. He leads the conservation biotechnology programs at Kings Park Science, curating the tissue culture and cryopreservation ex situ, which means outside its normal habitat for those who are not aware. Is that right?

Bryn Funnekotter: Yes

AM: Conservation, I’ll read that again, conservation collections. These specialized techniques are used to safeguard the future of some of Australia's most threatened plants. Bryn has worked in this role for the last two years, and was previously employed at Curtin University as a postdoctoral researcher in collaboration with King's Park Science, looking at how the cryopreservation processes, process sorry, damages the plants and how we can improve these cryopreservation protocols for greater survival in Australian flora. It's an absolute delight to have you here Bryn, and if I forget to do it at the end, I would like to say thank you for your work because we live in the most magical part of the world and our plants are amazing and the work that you're doing on this is, well, extremely important. So thank you. Would you please put your hands together for Bryn.

BF: Thank you Arlene. So welcome everyone, thanks for coming along to learn a little bit about my research and my work that I do at Kings Park. So very quickly, Kings Park and Botanic Garden, the Kings Park bit, was established in 1895, and it's about 400 hectares of space dedicated to the park. And more recently, in 1965, the Botanic Garden was officially established in Kings Park and we became Kings Park and Botanic Garden. So what's the difference between a park and a botanic garden? So botanic gardens have to actually document their collections of plants for the purposes of scientific research. So, the collection in our botanic garden is far more monitored. It's not just a random bunch of plants that I put there because they look pretty, they serve functions for conservation, for education and it's a big part of the work we do at Kings Park, is all the research associated with our botanic garden. We have about 3000 species in our botanic garden. So we are capturing about just around a quarter of the different species we have in Western Australia. So, while there's a lot of species there, there's a lot more that are not represented in our garden. All right, Kings Park science. So part of a botanic garden is the scientific research associated with it. Kings Park Science is world renowned, we are recognized as one of the leaders in quite a few spaces of plant restoration, ex situ conservation, our seed research is very important, as well as all the fire sciences and the management of our urban bushland. We’re very focused on how science can help us understand our flora as well as use it both for education as well as conserve it for future generations.

So Australia is very, very diverse. There's over 24,000 species of flora in Australia, 70% is endemic. This means they are not found anywhere else in the world, so Australia's flora is very unique. You won't find many of it somewhere else. And we have a lot of different climatic regions in Australia. So we get tropical regions, arid regions, semi-arid regions, Mediterranean regions, alpine regions, so we see a huge diversity of plants in all those different regions. Down here in Perth we are considered a biodiversity hotspot. So there is a lot of species focused around in this little area here. And a lot of them are under threat, so their native environment has been degraded over time. Obviously we have a huge amount of population in WA, just in this tiny little region here. So it's seen a lot of clearing. And what was there a couple hundred years ago is mostly gone. So that's why it's listed as a biodiversity hotspot, and a lot of the species are threatened in this area. So ex situ conservation, we have almost 1500 species listed as threatened. Basically, to be categorized as a threatened species, they have to have a low number of mature individuals, a reduction in the population size. So we would consider, sometimes there's just small amounts of species that doesn't spread quickly, it doesn't mean it's threatened. But if we see a reduction in that population size quite quickly, it can very quickly become threatened. So there's a key threatening process that is damaging that population. And if it's geographic distribution is precarious. So if it's sitting on a mine site and they've want to get rid of it because they want to mine up the soil, it does happen. But that species could be threatened at that point in time. So Australia has 35 flora listed as extinct at this point in time. They haven't been seen for a long time and there's records that basically say we should see these species in these locations, but we haven't seen them. We'll get to some interesting stories about some of the species we have in our tissue culture collection that were considered extinct, but some of the rest of them, the rest of the threatened classifications, the main ones we're interested in is the three, the critically endangered, the endangered and the vulnerable. These are all high priority species for us to conserve.

So around Australia there are 14 facilities, mostly the Botanic Gardens, working on ex situ conservation. One of the interesting things, so they will generally have one of these facilities, a seed bank for storing seeds of threatened species, cryostorage and micro propagations tissue culture, or a nursery production. So, some botanic gardens will just have a local collection of plants as a conservation collection. Here at Kings Park we're one of three that has the cryostorage capabilities. So there's only three places around Australia that are doing this work. The Australian Plant Bank and the Royal Botanic Gardens Victoria are the other two. Royal Botanic Gardens Victoria is very new with their cryo collection, so they're still in the process of actually preserving species. And the Plant Bank has been around a bit longer, but Kings Park was the first to start a cryogenic collection. Yep, okay. So seed banking, this is what we want to do, if we can, to conserve a species. So these are great. Like, if we can collect seeds and they're viable and they're good seeds, the seed bank is an excellent ex situ conservation option. They relatively easy to get a nice diverse genetic sample from a few different individuals in your collection, and they can last a very long time in the seed bank if they’re handled properly and processed properly. So Kings Park has been collecting seeds for more than 60 years now. We have around 13,000 collections. So we have a specialized seed cell that basically goes around the WA and collects seeds from all over Western Australia. And that's the main purpose, is to try and add many of our species into our seed bank. So we've collected over 4000 plant species into our seed bank. So that represents about just under a third of the Western Australian species are in our seed bank. So again, we still have a long way to go. But the work is, is very important. You'll find out later why we can't do this with some of our more specialized methods, like tissue culture and cryopreservation, because they take too long. Seeds are great, we can collect them, process them, and store them, and you can do hundreds of species in a year. So, if we can seed banking's our first ex situ conservation method.

But there's always exceptions, and not every species likes to produce nice seed that can be dried down and put into a seed bank at cold temperatures. So there's many species that don't produce enough seed. They basically propagate from rhizomes under the soil. Or there's issues with, some of our really threatened species where there's not enough individuals for nice cross pollination to occur, and they just don't produce very many seeds at all. We have issues where some species produce seed that are short lived in the seed bank. So even though you dry them down, you keep them nice and cold they still die off within five, ten years and what you're storing is dead seed. And that's not really useful to anyone. And some of them have real dormancy issues. So some of Australian species just the seed might be viable, but they just can't germinate or they won't germinate, we can't get them to germinate. So we don't completely understand the germination process and what key factors the seeds are looking for to start the germination. So a recent publication, and I was involved in this one, basically looked into what is considered these exceptional species. So it's a list of species that can't meet the conventional seed banking ex situ conservation programs we're looking to do. They identified 250 species basically from around Australia that we know are exceptional and we know what makes them difficult to put into ex situ conservation, but there's going to be many, many more to be added to this list. It was relatively recent, it happened in 2023, so it's only been a couple of years since we started classifying some of our more difficult species as exceptional. But one of the big things they did identify was that tissue culture is probably going to be one of the main methods for basically conserving these species.

So if we can't take seeds, so just quickly, tissue culture is the growth of plants aseptically on an artificial nutrient media. So there's little media and the jars we pass around, you'll see there's a little agar film, in that agar is all the nutrients the plant needs to grow. So it's a very flexible method, like we can change the nutrients we’re supplying to the plants in that agar, and we can get the plants to grow many different ways. The other thing that is quite useful with tissue culture is we can use a lot of different parts of the plants to initiate into tissue culture. So if we can't get seeds, we can take cuttings, we can take leaves, we can take flowers. Depending on the hormones that the plant growth regulators we give them, we can then force a flower to callus up and produce shoots or roots or something else. So it is very flexible and it gives us a lot more options for how we can start conserving these species that are difficult. It produces clonal plants. So you can see we’re chopping up plants in the bottom here. They are all clonal accessions. So we keep these clonal lines. So it is one of the disadvantages of tissue culture is it’s not particularly diverse the collections we get. So it's a lot more manual labor to maintain these collections as an ex situ collection. But we can produce a large number of plants from one single cutting, so we can keep multiplying them up until we have sufficient plants to go back into the wild and bolster a population where we are seeing declines in plant numbers, immature individuals.

So why do we use tissue culture at Kings Park? The main one is for our threatened species, particularly for when there's very small numbers of plants remaining in the wild. And this is normally when there's less than 50 plants remaining in the wild, and we see that quite often. It means we can go to each plant and take a cutting and initiate that cutting into tissue culture so we can conserve all the genetic diversity that is remaining in a population. It gives us a lot of control, lights, temperature, nutrients, everything we’re supplying to the plants, we can really baby the plants and get them growing as we want them to. So difficult to propagate species come under our list where they won't strike in cuttings, we can't get the seed to germinate, if there's other reasons why, if they're really sensitive to things like diseases. Tissue cultures are clean and sterile, so they're not exposed to diseases. So it's very useful that way round. Tissue culture is used by our orchid specialist as basically a very special way of introducing the mycorrhizal fungi to the orchid. So orchids require a fungal symbiotic association to grow, and that fungi is very specific to that orchid. So the issue is if the orchid seed germinates, it puts out small little roots, but they're not very well developed roots. They interact with the mycorrhizal network and they basically, the fungi provide nutrients back to the orchids and the orchids will continue growing at that point in time. If that fungi is not there the seed will germinate but it won't grow beyond a certain point, and it will eventually die off. So they, they use tissue culture because it means we can grow the fungi in tissue culture and we can grow the seeds and tissue culture and bring the right fungi and the right seeds together. Which is really important for some of our threatened orchid species.

One of the other ones that use our tissue culture facilities is plant development. So, plant development at King's Park are involved in trying to bring new species to the public, Western Australian species to the public forefront. They made the blue kangaroo paw. I don't know if many people know about this, but all the color forms of kangaroo paws that are out, our plant development team is involved in the crossings and the breeding of those different color lines. The nice thing with this is it's clonal, so you can basically produce lots of that plant that is producing that particular flower that you want or that morphology that you want.

We can change. So, the Kings Park Science tissue culture collection started in 1982. And the start was just can we grow the Australian species in tissue culture? So, a lot of the early collections and research that was done was basically taking plants from the botanical garden and saying, Can we grow them in tissue culture? What lighting conditions do you need? What nutrients do we need? How do different jar sizes affect them? How do different temperatures affect them? Like, so it was basically just trying to understand how Australian plants grow in tissue culture. Since that point in time, so I'll cover this first, I forgot. This is the process of tissue culture. So we'll go and collect our plant of interest, and one of the big main steps is disinfection. So any bacteria and fungi that are on the plant, our plant tissue culture media grows plants okay, but it grows bacteria and fungi a lot better. So you end up just with a nice culture of bacteria or fungi, and it's really gross. So one of the big bottlenecks of tissue culture is getting sterile, sterilizing all the microbial contamination off of your plant, and initiating into the culture conditions. So the initial work was, how do we grow our Australian plants and what media do they like? Once we get to the multiplication stage, we can start producing lots of those plants. And then we can change up the media, get them to start producing little roots in the agar. So, if you take a look through our plants here, you'll see pretty much none of them have roots on, the nutrients that they need will just diffuse up in the shoots. So they don't really require roots when they're in the tissue culture space. Some plants will naturally root, other ones are real pain and really difficult to get them to root and take them out of tissue culture. But the purpose of this is to provide plants that we can use for translocations. So the main goal is to say we can use tissue culture, we can take a cutting from one of the last remaining individuals and produce lots of plants that we can the return back into the wild.

So, once we had established that we can grow Australian plants in tissue culture, the focus shifted to really looking at threatened species, and exceptional species in need of conservation. So we currently have 46 species in our collection, 36 of them are threatened. And the rest are in there for research projects. We're doing some work with Alcoa on their tissue cultured plants that they use for post mining rehabilitation. And, we have just general plants that are used in our PhD students research projects. But the main focus is on these species of conservation concern. So, to chat about a few of the really nice species that have a nice story. I just picked a few. So this was one Grevillea scapigera that was presumed extinct. They basically hadn't seen the plant for, since this early collection in the 1900s until 1989 when someone stumbled across a population of six individuals. So the tissue culture lab was very new at this stage, and someone came along and said, they're not really doing well in the nursery can we try them in tissue culture? And it basically sort of kicked off the tissue culture program and it's use in the conservation space. So after a few years of growing in tissue culture and returning plants, there is a self-sustaining population growing near Corrigin. And there was a bunch of really interesting genetics work that was associated with this initial conservation collection. So I'll explain this for those that are not familiar with genetics. Again, this was not my area either. So, what we have here is the initial species that were found, the initial plants that were found. And the higher the number the more diverse that collection was. So those initial plants that were discovered had the highest amount of diversity, and they were all initiated into tissue culture as their own clonal lines. Over time the self-sustaining population kind of grew but what we find is they’re are all interbreeding, and actually we've lost a bit of that diversity that was initially there in the initial collection.

So another way of looking at this is in a 2D space. So this individual is very different from this individual over here, and the same as the opposite way around. So this was the initial collection and this was the first, oh no the second generation, the third generation, and then a few extra added into that third generation that they found. What you can see is that all the initial plants that were taken from the tissue culture collection are still more diverse than all the individuals that have basically cross-pollinated and produced individuals that basically represent, you know, the crossings of those two. So it's a great example of the fact that the plants are definitely crossing, we're getting new seed produced, that is new diversity, but it's not more diverse than that initial collection. So, the value of that initial collection is still very high for this species. But it did come with a few caveats along that line. This, the main finding was that finding appropriate sites was very important. So, they got grazed on if you put them back in the site and some kangaroos come along, they find it quite tasty and nibbbley as the fresh little shoots are there. So they had to do a bunch of work to make secure sites for the species. And a lot of volunteer work as well went into monitoring them, making sure they were weed free and pest free. But one of the findings was that having a genetically diverse ex situ collection is very important for the conservation of these species. But a good story, definitely very successful, and a great use of how tissue culture can be used for conservation.

So another one presumed to be extinct, which is why I like to point out that the 35 species listed as extinct could potentially still pop up eventually. Symonanthus bancroftii, so this was presumed to be extinct, last seen in the 1940s. A single male plant was discovered in 1997. I hope you can all see the problem with that, [laughter] Yep. It was initiated into tissue culture because we can at least multiply up the plant, but there's no way to make a self-sustaining population with a single male. They're all males at this point in time. But luckily a female plant was discovered one year later. So these two plants were brought together, seed was produced. It seeds quite well, actually. If you can have a male and female plant quite close to each other that they can basically produce seeds, and it really was a nice, easy little trial. The, the seed production area basically was established at Kings Park, which basically brought as many of the collections as we could together, to just produce as much seed as we could. And then they use that seed for translocation programs, to just introduce new plants back out into where we thought it should be. A bushfire near Corrigin basically stimulated the seal, the soil seed bank, and a whole bunch more actually popped up after that point in time. So we have about 35 accessions now of this species in the tissue culture collection. It is a fire ephemeral. So basically it pops back after a disturbance, particularly a fire event. It seeds and then dies off. So that's why you kind of..., it died off and then there was no disturbance event. And it was just something happened that made these couple pop back up, but a whole lot more pop back up after a fire. So this is quite commonly seen in the wheat belt area. So you get these very small, fragmented populations as the land got cleared to grow agricultural species. And the, the big fires that used to go through there became far less frequent. So some of these species that were threatened we know the key threatening process is actually a lack of fire in those small little fragmented populations that would stimulate these species. But you’ve got to be careful because it might be detrimental to other species, so it's a very careful thing that needs to be managed.

Androcalva adenothalia if you want to talk about plants that are not pretty at all. You know, quite often now, threatened species are these little clumps of sticks or something else. And they're not very cute. And it's hard to sometimes, you know, sell that as a, you know, everyone wants this in their garden because it produces nice flowers or something else like that. It produces teeny tiny little white flowers, in tissue culture, every now and again. They’re kind of cute, but even in tissue culture, it doesn't look nice. But there were two individual in 2005. So, tissue culture was used to just basically conserve those two individuals. They did die off in the wild. I can't find an actual report, but I know that the seed bank has about a few hundred seeds actually in their collection as well. So this species, when they find an appropriate translocation site and they know how best to manage that site, will go into a translocation program and there'll be work done on returning it back into the wild. The same, coming from the Wheatbelt area, it's potential that more of them might be discovered if a fire goes through an area, you might see them pop back up again. We don't really know. And that's the problem with a lot of our species, is we just don't know enough about them. So, the more we learn, the better it is for everyone. But we have this one in tissue culture because there was only two known on the particular site. So, definitely makes it worthwhile having us having this one in tissue culture.

Acacia volubilis, this one I'm working on right now. So, this was also one that was thought extinct. It was first collected in 1877, and then not found until 1996. So, this one's in Corrigan, which is like two hours out of Perth. You think you would have seen it, but quite often the way it’s where someone will be walking through an area and be like, ‘What is that? And yeah, that's all we need to sort of find some of our plants where we go. We haven't seen this one for, you know, quite, almost a hundred years, over a hundred years, and someone found a few individuals. There's about 100 plants in the wild when they actually went looking for it, in that spot. So there is a decent number out there, but it's still not in any means a lot of plants out in the wild. The issue, and it's quite strange for acacia, is it has very poor seed sets. So most acacias produce lots of seeds and they're very easy, you can collect seeds and you can bank them. So this species just really doesn't produce seeds, it flowers profusely and those flowers don't turn into anything. So, yep. And one of the other things we've had is the nursery took cuttings, to strike cuttings, to try and use them for translocation program. They've really struggled. I've taken cuttings and tried to put them in tissue culture and I've really struggled. So this is one of the advantages of tissue culture over just cuttings is I went through the process of (oops, I didn't mean to go forwards) somatic embryogenesis. So I can do horrible things to the plants, like making them just grow wound tissue and lots and lots of wound tissue. So callus tissue. And then I can change the hormone signals going to that tissue and say, I want you to be a shoot. I want you to be a root. So I've basically grown it up as a bunch of wound tissue. And then said, start forming little shoots. So I've now got tiny little shoots that are growing over there. It's over here as well, so you can take a look at it. It's quite interesting that it's got its baby little acacia leaves. So I took this from a mature individual. But forcing growth through this process basically takes it all the way back to, what is like a seed embryo. And it grows back its baby leaves and will hopefully mature leaves in a little bit. So this is looking like the method we're going to have to use to conserve this species. It is just not doing well through our nursery collection in comparison.

So one of the new other areas and always biosecurity risks are huge problems. Everyone's probably heard of the polyphagous shot hole borer and how difficult that is. Myrtle rust is on our list as another one that could be a huge problem here. So myrtle rust affects the Myrtaceae family. This includes your eucalyptus and your melaleucas and a bunch of the lilly pillies. So there's a whole bunch of Australian species that are very common around here that can be affected. It was introduced in 2010, in Australia on the east coast, and it basically spread all the way up from New South Wales into Queensland. It very much likes warm, wet conditions, so Queensland is terrible for it. And the issue is myrtle rust affects all the reproductive new growth. So when a flower starts to form, that's when myrtle rust really likes to grow. So what you had was species that would be very common, became very quickly weren’t producing seed. So they saw common species become critically endangered in ten years. So that's a huge change to the environment that happened over there. It was detected in the remote Kimberley up in the north in Western Australia about two years ago. It hasn't spread any further. It doesn't like to cross the arid semi-arid regions. But the south west is amenable to myrtle rust. So if it does get into the Margaret River region, Albany region, it could be quite devastating in that area.

So we're doing a bunch of work on some of the exceptional species we find up in the Kimberley region, mainly the lilly pillies. The Syzygium species. These basically produce really big seeds that are very, like, they just want to grow straight away, so they don't go through the dormancy stage. So tissue culture is basically a nice way of growing these plants. We’re just trying to develop protocols for as many of our Myrtaceae species as we can. So we just, if something happens to a common species we at least have protocols to start working on it. And tissue cultures, nice and clean. Myrtle rust is not going to grow in our tissue culture environment, they’re in their sealed jars. So that is one of the risks with the nursery collection. So even if we bring them down to Perth, they’re still at risk from myrtle rust which is spread as spores by wind. So once it starts spreading, it's going to get everywhere, which makes it pretty much impossible to stop it spreading once it gets to an area.

So yep, we basically have identified a whole list of species in urgent need of at least protocols for how to conserve them. Going into the, into the seed bank for what we can, or through tissue culture if we need to. Limitations of tissue culture. One of the big ones is this initiation. We can spend a lot of time just trying to get something to get into tissue culture if it is really covered in fungi and bacteria and it’s endophytic. So it’s in between all the cells. So as soon as we put them on to tissue culture media, that bacteria will multiply much quicker than the plant can grow. So that's the main issue. We do have a few slow multiplication poor rooting species. This is very much an optimization of the tissue culture process. It's one way basically we don't know enough about how these plants grow. So we don't know what to change in the tissue culture system to make them work that way around. But, we have a bunch of collaborations with other organizations around Australia working on these problems in the tissue culture space. Regular sub culturing, the plants just basically eat up the nutrients, as they grow so you need to replace that media every now and again. So this is where we get to our limitations for our current collection. There's myself and Katie, our technical officer, and we have to look after the whole collection. So, there's just not enough time for our collection to expand beyond that sort of 50 species. And if we were to maintain them indefinitely in tissue culture, we’re probably at our limit now. We wouldn't be able to add new things. So this is why we're going to do cryo, and I'm going to talk about cryo in a second. So really, tissue culture should be considered a short to medium term ex situ conservation technique. You should really do something with them in ten years that they in the collection, whether that's putting them back out into the wild or something else. But the other thing is to cryopreserve them.

So cryopreservation is the storage of germplasm at ultralow temperatures. So this is typically done in liquid nitrogen. It's very cold in liquid nitrogen, it's -196°C. So the advantage of putting something so cold is all the chemical reactions slow down more and more and more and more. At those temperatures, basically everything is in stasis. So as per the movies, you can cryopreserve someone and send them through space a thousand years and warm them back up, and they should be alive. We can't test that just yet, but we have cryopreserved quite a few species and things are looking pretty good so far. So we have a couple of Dewars at Kings Park, specifically for putting threatened species into cryogenic storage. So problem is, ice. So water does this horrible thing where it wants to form ice crystals, and it really makes my life difficult as a person trying to cryopreserve stuff. So as water molecules come together to form the ice crystal structure, the hexagonal phase, the space between the water molecules actually gets bigger. So that's why ice floats in water. There's more space in the thing. It makes it less dense than the water. And what that means is ice crystals basically expand. And if they expand in your cells, they just go pop and your cell falls apart. So, what we see when we start to look at ice damage in our samples is these big voids where the cells have just completely burst. So ice crystals have expanded, grown, and just completely damaged the tissue in those areas. So, they basically, can interfere with pretty much the whole cellular process. So it doesn't matter if they're small little crystals inside the cells. But as soon as they get big enough, they're really going to cause problems, and they, ice if they can form, if water can form ice, it will form ice if you try to transition them down into -200 degrees. So, it is a main challenge that we face trying to cryopreserve samples. One of the other weird things is if ice is outside the cell and you give it enough time, the ice crystals will grow outside the cell, get bigger and bigger, and suck all the water out of your cells. So you can get cells that just collapse in on themselves as the water moves out, as it gets bound into the ice crystals. And then to add to that, as soon as you take the ice crystals away and you try and regrow the plants, if the membranes are all folded in on each other, and you bring water back into that system, you can just pop the cells. So that's a pain or that you can get this complete loss of semi-permeability characteristics in your membrane. So, there's a lot of challenges with cryopreservation but it was my area of research in my PhD and my postdocs.

So we did do a lot of work looking into this. I really like Futurama, they nailed it. So if we can take water in the liquid phase and immediately put it in the glass phase, it doesn't give time for those ice, those water molecules to come together to form ice crystals. Physics gets in the way of doing that. It's a real pain. So physics would just let us completely cool something down. We can do that with tiny, tiny little droplets of water. They basically shoot droplets of water at very, very cold metal plates and they can go cool, we can do that. Like, that's not really practically useful for us. So one of the other things you can do is increase the amount of solutes in your cell. So water will interact with itself, but it also interacts with your membranes, it interacts with your proteins, it interacts with amino acids. So if that water molecule is already bound to something else, it won't be able to turn across and bind onto the ice crystal. So if we increase our solutes we can basically stop those water molecules coming together to form ice crystals. So what we use is basically what are called cryo protective agents. These are small, little chemicals that have a similar structure to water, and basically just bind up water molecules so that they're not available to form ice crystals. So you can see I froze some water here. And this is plant vitrification solution 2, this is one of our very common use of combination cryo protective agents. It doesn't expand, it has the same structure and it goes completely clear. So it looks basically like glass. So all those water molecules in there are basically stuck in their spot when you cool them down. The issue is it's toxic. So, we have to be careful getting the cryo protective agents into your cells to stop ice crystals, but also not kill them from the toxic side effects. So they're all like water, but they're not exactly the same shape as water. So they can denature proteins and membranes can fall apart if you start using them.

So there is a few stages of cryopreservation. The actual practical side of this is actually quite simple to do cryo. What we do is we take whatever germplasm we want, whether it’s seeds, if it’s tissue culture material, we can do it off fungi and bacteria. It's very commonly done. And we desiccate them to remove as much water as we can without damaging the cells in any way. We then chock them full of our cryoprotective agents, as much as we can, and then you can freeze them. You can throw them into liquid nitrogen. And if you carefully rewarm them, remove the toxic cryoprotectants, you can get nice growing plants out the other side. So this can be done in a day in the lab, and practically very simple. All these stages can be optimized and changed, and you can choose different cryo protective agents, you can choose different desiccation solutions. You can change your recovery media. You can change how quickly you cool them and warm them in liquid nitrogen. So as simple as it is, there's a lot of optimization that goes into this process. So cryopreservation began in 1992. The very first cryopreservation reported was in the 1960s on sperm. It was the late 80s before they really started to cryopreserve plants, and that was mainly focused on winter dormant buds. So they could take sticks with dormant buds of apple trees and throw them in liquid nitrogen, warm them back up and say, ‘oh look, that actually worked.’ It doesn't work with our Australian species because we don't get cold enough here to basically induce that dormant bud phase. But the work was done on the Grevillea scapigera, so as I said, it was a very important species for us and that collection is very important to us.

So the initial work was done on this threatened species, and had relatively okay success, using the very original methods that were out there. They didn't have a lot of mixes of cryoprotective agents. So we've done, there's been a huge amount of work into these cryoprotective agents, finding new ones that are less toxic or combinations that we can use that are more amenable to more species. The work that was done initially was very species specific, so one protocol for one species could not be used on another species. We’re starting to find combinations that are more broadly applicable to more species. So it is speeding things along as the research in this area has continued. So few tips. This is the tiny little growing portion of your tissue culture material, our primary source, our germplasm source that we put into cryogenic storage. We do this because it produces the best true to type plants. So, when you raise them again afterwards, they don't grow weird leaves or do other strange things. So they genetically, they're very similar to what you put in. So there's been a lot of genetic analysis in this space to make sure the cryopreservation process is not affecting your material. So we want to know that what we put in is exactly the same as what we get out, even if it's 100,000 years later.

So vitrification protocols have been very successful. We have 47 species now in cryogenic storage from our tissue culture collection. The other really big one is the orchid collection. So orchid seeds are teeny teeny tiny. They have very little energy reserves in that seed. So they’re one of the main ones that age quite quickly in the seed bank. So cryogenic storage just basically extends the lifespan of those seeds. (Oop, why did that go forwards?) So, a lot of the other interesting thing for the orchid collection is we can put the fungi in with those seeds as well, so you can grow the fungi on an agar plate and cryopreserve that at the same time. So there's over 3000 accessions collections of orchids in our in our cryogenic storage. Many of these are our really threatened orchid species. So we have a huge diversity of orchid species here in Western Australia and a lot of work that needs to be done on conservation in this space.

So a bit of stats. 50, just over a 50% survival. So what survival is is this one died, this one's growing, this one's growing, this one's dying, those ones are growing. Not every single shoot that we put in is actually going to grow. Especially when you get annoying species like Loxocarya cinerea, and I spent four years of my PhD developing that protocol for that species. We see such a huge range in how easy a species is to cryopreserve. So these are kangaroo paws, they're really great, I like them. The, some of our other species we really struggled to develop a protocol. So we do a lot of work in the fundamental requirements of cryopreservation. So what do cells need to do to actually start regenerating after cryopreservation? So 24 of the 37 species have survival rates greater than 30%. 30% is kind of like the standard cutoff. Like if you're above that, you can put a few vials into cryogenic storage and be pretty confident you're going to get something out that you can then reestablish your tissue culture lines and have plants that you can return to the wild.

So we definitely have quite a few on our list that need further optimization, future research. And there's a range of different cryopreservation protocols. I don't think I've got a slide here. So this is one of the things where keeping up with the latest in research is really important. So this droplet vitrification method is a modification of the very common method that was out there and what it does is significantly speed up the cooling and rewarming rates. So if you do get an ice crystal forming in your tissue, the faster you cool it down, the less time that ice crystal has to grow. So if we can speed up our methods, we can get significantly better survival rates at the other side. And this is a relatively simple change in the actual practical procedure but it wasn't implemented because, basically, they did a bunch of work in the 90s establishing the old vitrification method and then had a lapse in funding, in the 2000s, until we started up a new collaboration with Curtin University. And in that time this other method came out but was never implemented because they never had someone working in the research space. So it's been a good reason for us to continue working in the research space and spending time and effort, you know, working on understanding what's happening to our plants rather than just the pure, practical side of just putting stuff into cryogenic storage.

One of the other problems, genotypes. So these are the different clonal lines we have. We have some species where basically one plant does really well through preservation, and it can be the plant growing right next to it does really badly through cryopreservation with exactly the same process, same protocol. We don't always understand what causes this, but it's literally just something genetic or something wrong in the phenology of that plant where one will do really well through cryo and one won't. And these become really key areas for us to use in research. So we can start looking into the biochemistry and the biophysics of what's happening between the two, one that's doing well and one that's doing poorly in trying to understand what's causing this.

And we have some of these species, if you want a species that's full of water that's going to freeze and make a mess. We have two aquatic, carnivorous species. They're on our list to cryopreserve and they're going to be a real pain. We also have very, very hairy species. So this is the process of getting a shoot tip out of a hairy species where, you know, I'm not really sure what I'm looking at and what I'm cutting up and am I in the right spot? And this is a massively sped up video. But this will be just one of the individual shoot tips we're trying to cryopreserve, and you'll be looking to do close to 100 of them in a day. So, sometimes you get species like this, which are a nightmare to work on, and then you put it in the solutions and it floats on top of the solution. So that's, yeah, that's great. So fun ones where it's, it's going to be interesting to see what we can do with them, but we know they're going to be difficult.

So research into cryo injury, this basically starts our collaboration with Curtin University. They bringing in a lot more understanding what's happening to the plants, and we want to use that. So these are some plants regenerating. Why is that one growing so much quicker than that one? Yeah, we don't know. Yeah. But there's a whole range of factors from the problems with ice where most things stem, problems with membranes, the toxicity of the cryoprotectants, oxidative stress load. So you can see how blackened off some of the tissue gets after a while. So looking into these things and how we can optimize our protocols is really important.

Cell membranes. This was my early research work. They, since we’re basically, dehydrating our plants as much as possible, we run the risk of cell membranes coming together and forming a non bio-lipid by structure. And then when you rehydrate them, the membrane doesn't always split the way it's supposed to and you just made a hole in your cell. So, cell membranes are very sensitive to some of these effects of cryopreservation. We did a bunch of work into looking at the compositions. I'm going to skip some of this because it's very sciencey here. But what we saw was like, the phospholipids that are better at interacting with water molecules actually give us better cryopreservation results. And sterols are really important in plant membranes, they really stabilize membranes through stressful situations. But it was really interesting because they used this work to do some computer simulations. So this is a normal membrane and these are little water molecules, and how the water molecules interact with the membrane. This is when we start adding our cryoprotectants. So what happens is the cryoprotectants sit right where the phospholipid head groups are, so right into the membrane, and start spreading and stretching out your membrane. And they really change the characteristics of your membrane, and they’re nowhere near as stable when we start adding in our cryoprotective agents. So this is one of the issues of having molecules that are similar in structure to water, they kind of bind where water would normally be and start interfering with how they interact.

And this one is one of our main cryoprotectants, DMSO. And what happens is we're starting to completely fall apart here. So what we get is actually pores, struggling, there you go, pores forming in your membrane and you've lost all the semi-permeable characteristics of your membrane at this point in time. So this is one of our CPAs that we really want to kind of work on and try and replace with something less toxic. So this is a standard method. One of the things is this is all the steps where they find oxidative stress. So these are reactive oxygen species, things that basically mean your cell is not in balance and it's going to be struggling. So from isolation, getting your shoot tips, to desecrating them - that's no good, to putting them in the cryoprotective solutions - also not good, to recovering them. The acti-oxygens are not doing well. So it's basically kind of known now that the cryopreservation process induces a lot of oxidative stress on your samples, I mean, you've taken all the water out of them and thrown random chemicals into the cells, and they are going to struggle.

So we did a bunch of work when this, this was starting to really become a main thing in the cryopreservation space, looking at our antioxidant levels in our, in our plants. And what we're seeing is, this is in essence, like the cells have no antioxidant capacity left to regenerate and recover after cryopreservation. And we saw that in multiple species that we tested. But the nice thing is it's actually relatively easy to treat. So we can throw in some antioxidants in cells that are poorly performing can suddenly start regenerating a lot quicker. The recovery is much quicker. You can see they look way greener than the control ones. They look happier. So a relatively easy practical step to implement in our current cryopreservation protocol. So this is now becoming a standard thing we're starting to look at is just incorporate antioxidants into your cryopreservation protocols. Mitochondria. This is our current research. I hope you all remember that it's the powerhouse of the cell, important information. And we'll see how many people remember the citric acid cycle and the electron transport chain, and when we start doing this research you really actually... it's so complicated, Mitochondria. And so many different areas that can be affected by the cryopreservation process. We know that, visually, mitochondria can rupture, they can swell up and look really strange after cryopreservation, and you can lose this cristae density, which is basically the active site of the mitochondria. Basically, people have found that ADP, which is your energy source for your cells, is severely reduced by the cryopreservation process. So we went great, everyone had done that in sperm, they've done it in cancer cells. No one had done it in plants. So we're like, we really should start looking at what's happening to the mitochondria in plants. And they are doing horribly. So we started looking at the metabolic rate, the oxygen consumption rates of the cells, and they are horribly affected by the cryopreservation process. Our cells are really struggling in the early recovery phase. And this was one of our PhD students initial analysis into mitochondria. We had a whole bunch of questions of: Are they not getting oxygen and that's why they're not respiring? Are the cells dead? Are the mitochondria not functioning? Or is there just less mitochondria? So we have a whole bunch of questions where we actually, we know something is horribly affected, but we, we still don't know enough to really understand what's happening to the mitochondria during cryopreservation.

Some of the interesting stuff our current Honours student, who's just started his PhD did, was to look at the early recovery phase. And this was on a whole range of different recovery media. So we’re trying to give them things to regenerate. The control one, which has nothing. What you haven't read is the shoot tips that died, the metabolic rate of the shoot tips that died, and the blue ones are the ones that regenerated. So one of the common findings we saw is there's this huge lull period in the first 200 hours. And it’s seen in the one that did the best and in pretty much every single sample is something in the first 200 hours after cryopreservation significantly suppresses metabolic rate. So it doesn't matter if it does well or if it does badly, but in that early recovery phase is something we need to do to try and help mitochondria produce energy. And that energy is vital for the cells to start repairing, for cell cycles to start going again, and the plants to regenerate. So it's become a key area that we now starting to do a lot of research into.

I might, yeah, I might squeeze through this bit. Yeah. We’re going for a while. Yeah. I'll keep talking for a while. So if you thought just oxygen consumption was confusing enough, we could start breaking it down into many different areas of mitochondrial function. And I'm not going to go too far into this, but there's a few key ones that are really important, and it's the spare capacity in your mitochondria. So this is how much extra room the mitochondria has to respond to a stressful situation. And there's a few things like ATP linked respiration. So this is how much oxygen is going into producing energy for the cells. What we saw is as we increase our vitrification solution concentrations, the CPAs we putting into the cells, our spare capacity just falls off a cliff, basically. The plants basically have no ability to respond to any additional stress from this point of time onwards. And this is before we put them into liquid nitrogen. So, they are already stressed to the maximum from just the dehydration and the new chemicals we’re injecting into the plants. So yeah, it's, it's hard to know what we can do to try and limit the stress on the plants, we need those cryo protections in the cells but we certainly are stressing them significantly. And this was looking at the individual components. So there is two main ones, ethylene glycol and DMSO that are part of that mixture that seem to be the main cause of the stress.

Work still to be done. That's a lovely error bar for people in the science space. But we're certainly looking more into this as we're going along. So, we have a lot of work to do. There's a lot of species that are starting to show up as needing the specialized tissue culture and cryo protocols for conservation. We have a lot of work we want to start doing on the short-lived seeds. So the seed bank is 60 years old and they're starting to look into those initial collections and see which ones are doing well, which ones are doing poorly, which means we can start prioritizing species if they are poorly performing in the seed bank to go into cryogenic storage. There's a bunch of interesting things like pollen we can start putting in. So we don't have this currently in cryopreservation, but it's a great genetic source of genetic diversity. So if we can take pollen from male plants, and we have a single female, we can introduce a whole range of different male pollen to that female and get diversity that way around. Fern spores, no one's really done much work in the fern conservation of Western Australian ferns. But we have a few around here and a few threatened ones, so it is on our list. They can be very short lived as well in the seed bank, so it'll be an interesting one to do. And one of the things we're seeing is that the cryo protective agents we're using, while they have improved over time, there’s still hopefully alternatives that'll be far less toxic for our species. And the less toxic they are, the more applicable, applicable they'll be to a wider range of species.

So thank you very much. Thanks Eugenie, for your pictures and videos. Thanks to, the Australian Research Council, we've had quite a few grants that have covered the costs for this research space here. And yeah, I can leave it on that. I think I press too many times. Yeah, but. Yep. And a few of our students. So, Lily, Milana and Xavier that I presented some of their work. Thanks.

AM: Well thank you Bryn. There’s a lot going on behind this isn’t there. Wow, that was phenomenal, thankyou so much for sharing that. Are you happy to take some questions now?

So a quick question before this one. So the antioxidants really proves you should be eating blueberries? Is that right?

BF: Yep

Audience Question 1: That was excellent. I found that really fascinating, especially the cryopreservation work and I think the mitochondrial work is really exciting, actually. My name's Mark, I'm from Biosecurity and Department of Agricultural Fisheries & Forestry. I was, I'm actually a medical scientist as well, and we would regularly, and still do I believe, cryopreserve red blood cells in glycerol. Is that part of the solution that you use, do you use glycerol or is that a bit old now?

BF: Still a major component, and glycerol is great, it's not particularly toxic. So it is still one of the main components of the vitrification solutions. We haven't really found an alternative that's better. But the combination of different cryoprotective agents can be sort of, they can almost be synergistic as they become less toxic as you use more of them. So they are getting more complicated with more components as we’re going along? Red blood cells are great because they are small, and diffusion into them is quite good. Compared to something more complicated, like shoots. Audience member 1: Yeah, and there’s a lot of them. Also, it's not directly related to your work, but actually, this is. Grevillea, particularly I'm talking about now, when you have such few number of species, so few individuals, when they’re cross breeding the more that you propagate I’m assuming that the less diverse they become? So that would be a problem I imagine, it would put them at a much greater risk of a disease or something like that. It would wipe out the whole species?

BF: Oh, yes. Very much, very much so. But that doesn't mean the conservation effort shouldn't be done at the same time. So they are certainly at risk of what is, in essence, in breeding. And what you get with those very small populations is lots of non-viable seed, or yeah, susceptibility to more diseases or stunted growth or strange growth forms coming out of them occasionally.

Audience member 1: Do you crossbreed with other species, other similar species?

BF: No, not for the conservation collections. We’re very much focused on maintaining a true to type collection of that species. Plant development is far more interested in those crosses for disease resistance or better growth forms. But they are not going back out as a conservation collection, they're growing as a display plant.

Audience member 1: Great. So and one last question to biosecurity. Is the collection at Kings Park purely native species, or do you have foreign or introduced species?

BF: We have a few, for research purposes purely. The collections are Western Australian species.

Audience member 1: Do you have much of a problem with foreign species from the community, within the environment, invading Kings Park? Is that much of a problem for you?

BF: Yeah, yeah, we have plenty of grasses that shouldn’t be in Kings Park. And yeah, they can be huge issues for how they affect fire severity, stuff like that. So yeah, certainly an issue. But not yeah, they're not they, they're in the environment and they can be managed in small areas. But for the wider W.A. flora, we just kind of have to live with it.

Audience member 1: Thank you. That was excellent.

Audience Question 2: I was just wanted to know where we could get the information for the 46 species that you are trying to revive? It would just be interesting to know which species.

BF: So the department as a whole has a bunch of regional offices that are involved in the management of threatened species. What we classify as threatened species. So it's definitely a department wide collaboration on that work. So Kensington has the threatened species seed collection. If they're having issues they will quite often raise it to us to say this species is being particularly difficult, can we try tissue culture? Or if one of the regional offices says, I found this strange plant and we don't know what it is, quite often they will do collections at that point of time, and some go to a herbarium for identification and some might come to us to try and strike as a nursery collection or a tissue culture collection. So really depends on what's found out there. But it's more that we can all work together to conserve a species if it's something new. Or if something gets flagged as in need of conservation, that way we are available to help in that space. 

Audience Question 3: How do you tell the difference between female and male?

BF: Basically how the flower forms. So you get your anthers or your pistols on the male or female plants. So the, the flowers look a bit different between the male and female ones. A bit like humans as well, they look a bit different.

Audience Question 4: I'm just wondering if there's been any research on the long term effects on the resilience of the plants with the cryopreservation.

BF: There's been genetic analysis that basically says they look genetically exactly the same. For the most part, the finding is you might get a little bit of epigenetic changes. So, that changes how the plant might look a little bit. But they are very, they're not massively affected genetically by the cryopreservation process. So it does seem to be quite good that way around. And we've never seen really weird things coming out of cryo yet, but hopefully we don't.

Audience Question 5: The video you showed, Futurama, love it. But just something I think about, was thinking about when you were talking is that was basically uninterrupted power and resources to keep that tank going. What's the future proofing you do to try to get all of that research and work to last like 1000 years.

BF: So, yeah. So it is it is an issue for long term collections, that you have a consistent liquid nitrogen supply. So very important. One of the other things is good record keeping. So the plants are going to be in there for at least 100 years, hopefully. So one of the things we found with the early stuff that was put into cryo is someone will just write a number on it, and they're supposed to link to a database that no one has access to anymore. It can certainly be an issue with making sure we're thinking in terms of centuries about how we are managing this collection. It is an issue in I know there's a lot of tropical countries that wanted to do cryopreservation. They have a lot of species that don't produce seed that can be seed banked. So cryopreservation is their long term conservation option. And getting access to a consistent liquid nitrogen supply is the main limiting factor for them. So they can get small amounts, and do some research, but they can't get big enough amounts to actually conserve a collection. So here I think we're quite fortunate with the resources we have and the policies in place for conserving our threatened species that they will be safe for centuries. But certainly, yeah, management of the data associated with the collection has been raised as a potential, a potential risk to our collection. Yeah. 

Audience Question 6: I was just wondering how specifically you disinfect the samples before growing in the Agar and can it vary between plants without damaging them?

BF: Yep. Mostly around the bleach molecules, they quite good. So there is quite a few different kinds of bleach you can use. And plants are quite resistant to bleach. So it is our normal starting point. We can use antibiotics, we can use antifungal agents, we can do horrible things like mercuric chloride which fungi hate and plants can just survive on. So there is quite a range of options in the sterilizing space. We need to be careful with the antibiotics because resistance issues are showing up more and more and more. But most of the time we can get something successful, using one of the bleach family of chemicals. Okay. And second question before we let me work, and I. Audience Question 7: I was going to ask when your last choice is cryopreservation and you can’t cryopreserve, what is the last thing you do?

BF: After cryo?

Audience member 7: If you can’t cryopreserve?

BF: We have a few in our collection that haven't been successful yet and they basically become our research focused species. So if we are struggling with one, we palm it off into understanding the fundamentals of what's happening in those plants. So we'll start looking into what's happening with water. What's happening with antioxidants. What's happening with cell membranes? So until we can then develop a successful protocol they become a research focus collection rather than an ex situ collection.

AM: Any other questions before we let Bryn go home to his family, and you to yours obviously. Bryn thank you, that was wonderful. There were a couple of points in your presentation where I just got a sense that these are your botanical babies, the way that you described them and I felt some absolute love for them. So thank you for sharing that part of it too because we all know that science is complex and you need patience and you got all of that in there but I just love the fact that these are really important and they’re really important for all of us. And another reminder for me that nature really is the boss and we have to remember that sometimes, don’t we. Will you please give Bryn a round of applause. Fantastic presentation, thank you for your time.