Suspended Gametophyte Inductions - Success Stories?
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Hi everyone!
I'm curious if anyone has had any luck inducing gametogenesis + successful fertilization in gametophytes in suspended culture? Embyronic sporophytes are much more successful in direct seeding trials (You're not gambling on successful reproduction/development occurring in the field conditions), but inducing in plates/trays and scraping biomass is extremely space-inefficient and doesn't scale well.
Ideally, we need to be able to change conditions of a high-density gametophyte culture (Or high volume, low-density) and achieve high-% induction to yield any meaningful amount of sporophytes if direct seeding is to scale-up. However, simply turning on white light on a black tank of gametophytes hasn't worked for most folks I've talked with, myself included. Light, density, and tank diameter all intensely interact with the light distribution in a tank (>99.9% blue light is absorbed in less than 1cm of my culture tanks...) and I expect the uneven lighting to be the major contributor here.
I want to check whether other folks have already achieved or optimized this before I spend too much time and energy trying to tackle it. If you have- What worked for you and your species?
Currently working on Bull Kelp (Nereocystis) and Giant Kelp (Macrocystis), but interested in any kelp species you've had success in!
@maisie_musor1057 tagging a few people who may have relevant experience and perspective: @margaret_aydlett @javier_infante @rebecca_lawton @ryan_marchington1903
@maisie_musor1057 Hi Masie, we've actually just finished some experiments trying to answer this question. We're working on Ecklonia radiata and have been testing the effects of stocking density (0.1, 05, 1 and 5 g FW/L), light intensity (5, 20 and 60 umol photons/m2/sec) and light colour (red vs white) on gametophyte growth and sporophyte formation. We measured these parameters after 2 and 4 weeks.
I haven't had a chance to statistically analyse the data yet, but based on eyeballing some graphs there looked to be interactions between the 3 factors (no surprises there!). No sporophytes formed in any of the white light cultures, regardless of stocking density or light intensity. Sporophyte transition (% gametophytes that formed at least one sporophyte) was highest under the 20 umol light intensity, regardless of light intensities, and was comparable at this intensity under stocking densities of 0.1, 0.5 and 1 g FW/L, but was much lower under the 5 g FW/L density. In general, biomass productivity (total g DW biomass produced per culture) was comparable or higher in red light compared to white light for the same stocking densities and light intensities, and a light intensity of 20 umol outperformed the other light intensities under the sane stocking densities and light colour. Overall, biomass productivity was highest with a stocking density of 5 g FW/L, 20 umol and red light.
These experiments were run in free floating cultures (circulated by gentle aeration) in 250 mL plastic containers. We had to use this size so that we could fit in the required number of replicates to test all the different treatment combinations and time periods into the growth cabinet. Even though these cultures are a small volume, I still believe they provide a reasonable approximation of the conditions that would be experienced by gametophytes in a larger scale culture, and results are therefore more relevant/scalable than would be obtained if we tested these parameters in static/attached cultures. We will still need to do a small amount of optimisiation when we scale up to e.g. a carboy of gametophytes for inducing, but these results give us a starting position to fine tune from. They also highlight which factors are key to consider for growth vs induction.
Happy to chat in more detail/meet online about this experiment or any of our other work if that would be useful - just send me an email on rebecca.lawton@waikato.ac.nz.
Good luck!
@rebecca_lawton Thanks for such a thoughtful reply, I feel like I've talked with several labs and nurseries who really want to achieve this but doing so reliably has been challenging, so its interesting and exciting to hear this! I ended up doing some more light modelling work against predicted gametophyte reproduction and am finding similar predictions for my species, so I'll have to give this a test! I'd love to chat about it and will reach out
@maisie_musor1057 I just realized there was a pretty big typo in my first reply to you - I said no sporophytes formed in the white light cultures, but this is wrong. No sporophytes formed in the red light cultures, they only formed in the white light cultures. Sorry about that!
@rebecca_lawton Thanks for such a thoughtful reply, I feel like I've talked with several labs and nurseries who really want to achieve this but doing so reliably has been challenging, so its interesting and exciting to hear this! I ended up doing some more light modelling work against predicted gametophyte reproduction and am finding similar predictions for my species, so I'll have to give this a test! I'd love to chat about it and will reach out
@maisie_musor1057 interesting subject, questions and replies.
I'm interested in this method for using a robotic seeder for deployment of gametophyte directly on seedlings.
And deployment new methods for seeding and harvesting.
Following
Craig
@craig_lewis Thanks Craig, nice to hear! I am similarly interested. In the short term, to facilitate scale-up of direct seeding approaches and technology in kelp restoration in BC, and eventually to develop a nursery in the Canadian Maritimes or New England to stabilize high-quality seed production and hopefully avoid the challenges and limitations of seed string. Would love to keep in touch
Much to research and sort out in the meantime...
@maisie_musor1057 in our facility we have found seed string has alot more headspace that we once assumed--and has proven much more reliable and productive than other methods. Obviously there are some limitations to scale but I wonder how far off they are. Would be curious to hear what you see as the critical bottlenecks of seed string production.
@toby_sheppardbloch in an aquaculture context, I totally agree. Seeding gametophytes onto seed string goes a long way, and is fairly reliable and productive for the current scale of aquaculture here. However, building a sporophyte nursery is expensive, seed string is expensive, and seed string can often break on deployment. Currently, adhesive-binders for direct seeding are too variable in efficacy and have lower productivity than seed string, but a lot of people are working to improve them and some are starting to have success. Once there are improvements that stabilize the reliability, direct seeding could give a lot of flexibility to more remote farms and nurseries because you can completely eliminate the costs associated with the sporophyte nursery and seed string. I think seed string and direct seeding have the potential to be quite complementary.
More immediately for my work though, restoration has different constraints than aquaculture. Seed string is vastly more space efficient in the nursery than green gravel (especially for buoyant species that require large cobbles), but we are trying to re-establish kelp beds and deploying lines directly onto the ocean floor is not really ideal or the solution we are looking for. Green gravel works okay if you're doing targeted, small-scale restoration, but creating affordable infrastructure to grow it, transport it, and deploy it in places like BC where restoration is remote just isn't feasible. That, and its just a lot of work to be lugging around rocks!
Currently, I'm able to produce on the order of hundreds of grams of gametophytes in wine-fridge incubators with minimal infrastructure, and am continuing to develop a method to essentially broadcast disperse that biomass directly to the ocean floor. However, establishment of that seed onto the ocean floor is likely improved if sporophytes are already induced (not gambling densities and env. conditions for reproduction, and giving growth a head start), but controlling the induction hundreds of grams of biomass is challenging. I've been doing this in large glass baking trays at present, but its not space efficient and can only support a fraction of that biomass. It would be ideal if we could just dilute our gametophytes into a larger column tank, turn on white light, and achieve efficient induction of suspended seed. Unfortunately it's quite finicky and easier said than done!
Happy to chat any time about all these types of things! And maybe see you all at ISS? Hosted in Victoria this year where I am!
@craig_lewis Thanks Craig, nice to hear! I am similarly interested. In the short term, to facilitate scale-up of direct seeding approaches and technology in kelp restoration in BC, and eventually to develop a nursery in the Canadian Maritimes or New England to stabilize high-quality seed production and hopefully avoid the challenges and limitations of seed string. Would love to keep in touch
Much to research and sort out in the meantime...
@maisie_musor1057 I just realized there was a pretty big typo in my first reply to you - I said no sporophytes formed in the white light cultures, but this is wrong. No sporophytes formed in the red light cultures, they only formed in the white light cultures. Sorry about that!
@maisie_musor1057 in our facility we have found seed string has alot more headspace that we once assumed--and has proven much more reliable and productive than other methods. Obviously there are some limitations to scale but I wonder how far off they are. Would be curious to hear what you see as the critical bottlenecks of seed string production.
@toby_sheppardbloch in an aquaculture context, I totally agree. Seeding gametophytes onto seed string goes a long way, and is fairly reliable and productive for the current scale of aquaculture here. However, building a sporophyte nursery is expensive, seed string is expensive, and seed string can often break on deployment. Currently, adhesive-binders for direct seeding are too variable in efficacy and have lower productivity than seed string, but a lot of people are working to improve them and some are starting to have success. Once there are improvements that stabilize the reliability, direct seeding could give a lot of flexibility to more remote farms and nurseries because you can completely eliminate the costs associated with the sporophyte nursery and seed string. I think seed string and direct seeding have the potential to be quite complementary.
More immediately for my work though, restoration has different constraints than aquaculture. Seed string is vastly more space efficient in the nursery than green gravel (especially for buoyant species that require large cobbles), but we are trying to re-establish kelp beds and deploying lines directly onto the ocean floor is not really ideal or the solution we are looking for. Green gravel works okay if you're doing targeted, small-scale restoration, but creating affordable infrastructure to grow it, transport it, and deploy it in places like BC where restoration is remote just isn't feasible. That, and its just a lot of work to be lugging around rocks!
Currently, I'm able to produce on the order of hundreds of grams of gametophytes in wine-fridge incubators with minimal infrastructure, and am continuing to develop a method to essentially broadcast disperse that biomass directly to the ocean floor. However, establishment of that seed onto the ocean floor is likely improved if sporophytes are already induced (not gambling densities and env. conditions for reproduction, and giving growth a head start), but controlling the induction hundreds of grams of biomass is challenging. I've been doing this in large glass baking trays at present, but its not space efficient and can only support a fraction of that biomass. It would be ideal if we could just dilute our gametophytes into a larger column tank, turn on white light, and achieve efficient induction of suspended seed. Unfortunately it's quite finicky and easier said than done!
Happy to chat any time about all these types of things! And maybe see you all at ISS? Hosted in Victoria this year where I am!
@maisie_roymusor Hi Maisie! I've done a lot of inductions for Giant Kelp and recommend the following:
I recommend using a low gametophyte density because it influences the reproductivity a lot. The sporophytes will usually form within 7 to 14 days.
If you need more information on exact densities, light parameters, temperature parameters, nutrients and other additions to speed up and enhance induction you can send me an email.
Tristan S (TristanSteengroef@outlook.com)