Neos from seed - a DIY adventure

[myxodex]

I've decided to give some details here as one of the most frustrating things has been accessing information. As I've lost functionality of the manual functions on my basic camera the few photos I share are of poor quality ... hopefully I can fix this in updates to this thread.

About 7 or 8 years ago (IIRC) my Neo. richardsiana self pollinated and developed pods. This triggered my imagination and I started wondering which of my 40 odd neos might make interesting crosses. I had also read that neos often don't breed true, and that some of our varieties emerged as seedlings from selfings; such as Sengaku from Tamakongo. This didn't put me off, instead it made the project seem even a bit more interesting, bit of random genetic chaos in the outcome ... bring it on ... I can live with that.

At first I didn't consider doing it at home, I made a Hanagoromo x Hien cross and sent this together with some richardsiana pods to a seeding service. This didn't turn out to be successful and the person I communicated with said that neos can be tricky, he got germination but ultimately no seedlings.

I decided to make up my own medium and do it at home. I make my own fertiliser and so I already had all the salts, I just needed some form of peptone and some agar. I also had written a script (computer program) in R for turning recipes into ppm for individual ions, so tedious calculations weren't an issue, I just needed to convert the output to millimolar (which is how propagation media are presented in papers). The question was where to start.

I came across a recommendation of P723 for Vandas. P723 is approximately 1/4 strength Murishage and Skoog (MS) medium. So I made up a few variations of this basic formulation and sowed some seed. I got germination but the protocorms went transparent, like tiny green spindle shaped emeralds. This looked like vitrification (aka hyperhydricity) and it's cause is debated, but of a number of candidate causes, the one that seems most reproduceable is NH4 or ammonia toxicity. I needed to do more research.

After much searching I came across a paper on Neo seed propagation written in a Korean journal. The paper was in Korean, but it did have an English abstract. This was a start at least. They used a Japanese fertiliser called Hyponex. Hyponex comes in a number of versions and the company do not give any details beyond the NPK. More googling and I found some numbers; one of the most commonly used in propagation media has NPK 6.5-6-19 of which 15% of total N is NH4. The recipes in grams per litre were as follows;

For seed sowing; Hyponex - 3g, Peptone - 4g, Sucrose - 30g, agar - 10g ... adjust to pH 4.5
Replate medium; Hyponex - 1g, Peptone - 2g, Sucrose - 60g, NAA - 1 mg, agar - 8g, ... adjust to pH 5.0

I ended up making some media with NH4 at 15% and 30% of total N and at pH 4.5, 5.0 and 5.5. I got germination on both the 15 and 30 % NH4 media and at pH 5.0 and 5.5, but not at pH 4.5. I used a mix of organic acids in my medium, added as salts of NH4 and K. This was to serve two functions, firstly to provide some buffering at low pHs (MES is useless below pH 5.5) and secondly organic acids can help protect against NH4 toxicity. All the NH4 was added as organic acid salts. This proved to be a successful approach.

The nice thing about neos is that their pods ripen at 5-6 months and the seeds germinate within three weeks, indeed generally you can tell in the second week, ... so they are quick out of the blocks.

Here a pic of some seeds at about 3 weeks after sowing.

 

[myxodex]

Oops no pic
It's been so long since I posted I have to figure out how to get Flickr links to work ?

 

[myxodex]

... another try

 

[myxodex]

[/url]Germinat by Tim Dexter, on Flickr[/img]

 

[myxodex]

Finally

Anyway, after 12 to 18 months, depending on the cross you get these :

[/url]ToyoXHien by Tim Dexter, on Flickr[/img]

which are ready to replate. Unfortunately this is where I've been stuck for some time trying a bunch of medium recipes for replating.

 

[myxodex]

The best medium so far is the most tedious to make up (it just had to be). These are Hien x Manjushage and it begins to look like I've finally found a replate medium that works.

[/url]HxMonSWM by Tim Dexter, on Flickr[/img]

 

[myxodex]

I can post details of these media if anyone is interested.

 

[Happypaphy7]

Thank you for posting detailed info!
I am interested in knowing more.

So what's the time lapse between the last two photos?

Is it typical that you use different media for replates?
Why do you think the recipe used for other orchids didn't work for neos?

I think neos (I think most others) are grown in flasks for as long as possible before deflasking. I think I read even 3- 4 years even. Then they get potted up in sphag and go directly into the shops for quick sale or grown at nurseries depending on varieties and purposes.

Which lab have you tried?
I think Troy Meyer germinated and sold flasks of Amami type a few years ago.
Then some neo cross on his website states no germination or other failure.

I have five pods developing on three different neos. Some of these are selfing and others are crossing of different varieties.
I'm amazed at how fast these pods grow big!
I will have to find good lab who is experienced with neos.

 

[SlipperFan]

Those are amazing roots!

 

[TyroneGenade]

I am also interested in knowing the medium formulation. You should publish it in an orchid journal.

 

[abax]

Congratulations! This is exciting to see. I hope you'll
try to keep us posted as your plants grow.

 

[myxodex]

I will go into more details in subsequent posts and try to answer some of the questions. But first a bit of background. I did this ultimately because it was a challenge and because I wanted to see the process for myself. I don't have a breeding plan as such. I do have a curiousity about the origin of all the diversity in the neo varieties; such as how much is down to mutation alone and how much is down to a large diversity in the neo populations before the Japanese started collecting and growing them. Obviously I won't be able to answer any questions about the diversity of this species, but it piqued my curiousity as to how varietal crosses pan out. Either way it's been rewarding, but very frustrating; tenacity and patience are definately required for anyone thinking of doing this at home.

Although I've got some of my ingredients from scientific suppliers (so laboratory grade) not all of my ingredients are. In the UK most scientific companies will not supply to individuals and this is a nuisance. This does introduce a problem with interpretation of my results, although this is more likely an issue with macronutrients rather than micronutrients. Companies that produce media for commercial use do batch test all their ingredients against phytotoxic properties. How important this factor has been for me I cannot know. Also my situation doing this at home also places limitations on when I can do this. When OH is away on one of her trips to OS meetings/events, a few times a year, I take over the kitchen and adjoining room. She doesn't have an issue with this, but wouldn't like the inconvenience, limited kitchen access or the smell of IPA (rubbing alcohol) being sprayed on liberally on this and that. Having said that, I have solved a few problems along the way which might be of use to anyone else crazy enough to DIY this.

If anyone knows if and how I could post tables, say spreadsheet type, in a post that would be cool, but I guess it is a bit outside what this forum is about.
It's just that a table allows one to compare different media systematically, and I thought a table showing some standard media and some of the formulations I've used, could be helpful.

 

[Happypaphy7]

With regard to mutation, I think neos have great ability to mutate into different "varieties".
When I read some of the plants on Korean websites, nearly all the new varieties in the last two decades or so carry descriptions like "...this one arose among the selfing of Amami variety..." Then some from seed propagation ( not sure selfing or outcrossing) already known varieties like Shutennou, Tamakongo, Setsuzan... and others are from crossing two different known varieties.
Mutations seem more common among Amami selfings than others based on what I gather.

 

[myxodex]

@Happypaphy7. I will get back of the issues you address in your last post later. For now I will try to answer some of the questions in your previous post.

The time between the last two photos was about five minutes, ... because they are different crosses seeded about a year apart.
The second to last is Toyozakura X Hien and is about 14 months after seeding. The thing is when they reach this stage they slow down because they've exhausted most of the nutrients and eventually they will loose vigour. This means that they haven't changed a lot in the last few months and I don't have any of the good replate medium, but I will make up some more when I get an opportunity in August (OH away). The last photo is of Hien X Manjushage and is about 2 years + few months; they are the second batch replated from the mother flask 8 months apart which means they have been rather stationary for a long time and yet they did respond to replating, which is lucky as loss of vigour from tardiness in replating can be the difference between success and failure. If I'd replated onto the good medium sooner they would probably be more advanced than this. So basically I'm confident that if I had more control over timing, this whole process would go much quicker. It will all be a lot easier when I have optimised my formulations for replate medium because I've been through a lot of unsuccessful formulations. I only had 6 flasks of the good recipe to begin with.

It is often the case that a different replate medium is required. The conditions for germination and development (growing leaves and roots) can be different. My neos die within a few months if I plate them onto germination medium. I'm seeing two major issues here; (1) NH4 levels and (2) osmolarity of the medium. When the replate medium has the same levels of NH4 as the germination medium (3.6 mM) the roots die off first and turn black at the tips and the plants vitrify (or go glassy), but if the NH4 is too low in the replate medium root growth dominates and shoot growth is poor. Unfortunately it seems that different crosses have different NH4 tolerances and I might end up needing different replate media (NH4 levels) for the different crosses. This issue has also been found for phals, and again different NH4/NO3 optima can be needed for different hybrid strains. In the recipes from the Chung paper that I listed above you will notice that the sucrose is increased from 30g/l in the germination medium to 60g/l in the replate medium. It is said by some in the literature that anything above 20g/l sucrose in media formulations is beyond the energy needs of the plants and is all about getting the correct osmolarity. It seems that epiphytes more typically need increased osmolarity for development. You might then ask how come my plants begin development in the mother flask ? Firstly I plate out quite a lawn of seeds mostly, and the first to develop are those that are pushed up away from the medium ... how they survive like this is a mystery. Secondly my venting system is not ideal as it allows the medium to slowly dry out increasing the osmolarity. The issue with seeding density might relate to NH4 levels as well; one of the crosses I accidentally sowed one quite densely and the other sparsely, the sparse seed plate did not do well, so I'm thinking of further reducing the NH4 in my germination medium as I suspect this is a key variable for neos.

As to the issue with the "standard" media. There are a bunch of media derived from, or based on Murishage and Skoog (MS), such as P668 and P723.
MS, P668 and P723 have roughly 20, 10 and 5 mM NH4 respectively and these are IMO way too high for neos. These media work quite well for a number of orchids, and bar some exceptions, most papers I read on propagation of Phals, Vandas and some other epiphytes have seen some difficulty with these type of formulations. There are quite range of newer mediums out there, and I suspect that academia might be a bit behind the curve a bit on this and that commercial setups are using their own media formulations which are not shared. I have tried to get some specific information in correspondence with one seed sowing service about their media and the answer was "in house recipes". There are companies that will make up medium to your specifications and some might use this route. In short there is a lot of commercial secrecy out there and it's understandable. This is one motivation for me to provide details here.

Finally as to seed sowing services and growing neos, apart from the considerations in the previous paragraph, there is another factor. I've already seen how some crosses like Koukakuden x Hien seem to be very vigorous, while others are slow and fragile. So I suspect that the more "wild type" varieties and crosses will be much easier to propagate and have higher tolerances to different media formulations than some of the more "difficult" varieties. This is why I'm not doing selfings at this point. The seed sowing guy I was in contact with said that neo selfings are really difficult ... it stands to reason. Selfings have a greater potential to uncover silent mutations by loss of heterozygosity in seedlings. If there are a lot of heterozygotic mutations that weaken a plant when they become homozygous then the result will be a lot of weak seedlings.

 

[Happypaphy7]

Thanks.
I wish I had a facility & enough space to do the lab work on my own.
I thought about sending my seed pods to Korea but then in the wild lucky incidents of any good mutation like leaf variegation, I wouldn't trust they would even let me know or share with me. Pressure for money & fame is overwhelming over there. lol

Regarding selfing and genetic issues, i understand things follows the inheritance rules, but some selfings seem rather vigorous.
Kinyuko, I think was a mutation from a typical bean leaf crossing, which is very odd, and it is reported that it was so slow that the it took three decades to bake five divisions. After that, obviously it was selfed and it has become quite common in the market. What they say is that these numerous selfings are a lot more vigorous than the original mother plant.
I'm not sure some are mericlones of these vigorous selfings, or sib crossing ( still inbreeding, isn't it??) of these selfings.
So I guess initial selfing or inbreeding can yield some positive results?

 

[myxodex]

Thanks.
I wish I had a facility & enough space to do the lab work on my own.
I thought about sending my seed pods to Korea but then in the wild lucky incidents of any good mutation like leaf variegation, I wouldn't trust they would even let me know or share with me. Pressure for money & fame is overwhelming over there. lol

Regarding selfing and genetic issues, i understand things follows the inheritance rules, but some selfings seem rather vigorous.
Kinyuko, I think was a mutation from a typical bean leaf crossing, which is very odd, and it is reported that it was so slow that the it took three decades to bake five divisions. After that, obviously it was selfed and it has become quite common in the market. What they say is that these numerous selfings are a lot more vigorous than the original mother plant.
I'm not sure some are mericlones of these vigorous selfings, or sib crossing ( still inbreeding, isn't it??) of these selfings.
So I guess initial selfing or inbreeding can yield some positive results?

Yes, it is difficult to do this at home. Also I think you may well be right in that it could be that you don't get the best of the seedlings from a seeding service.

You are right about seedlings having more vigour, partly I guess due to selection. My Manjushage seedling grows like a weed; it's now a big 20+ growth plant.

 

[NYEric]

 

[TyroneGenade]

If anyone knows if and how I could post tables.

In the grand scheme of things that would be ideal but I think for those of us that would try this ourselves we only need the concentrations of the stock solutions and the final concentrations for each chemical.

 

[myxodex]

some details - neo germination medium - neo.oa25

My current germination medium (25% N as NH4) is working well. I have a reduction in the NH4, K and organic acids planned for a new version at about 20% NH4. It's way too complicated and I'm looking to simplify this, but this is how I make this medium atm, and it works very well.

The concentrations of the individual ions are as follows;

macros in millimolar :- NH4 [ 3.6 ] , NO3 [ 10.6 ] , PO4 [ 0.9 ], K [ 11.9 ], Na [ 0.1 ], Ca [1.95 ], Mg [1.0],
...................................SO4 [ 0.9 ] , Cl [ 0.28 ], succinate [ 1.5 ], L- malate [1.3 ], citrate [ 0.9 ], oxoglutarate [ 1.2 ]

micros in micromolar :- BO3 [ 29 ] , MoO4 [ 0.4 ], Fe [ 48 ], Mn [ 54 ], Zn [ 20 ], Cu [ 0.08 ], Ni [ 0.04]

vitamins + aas [mg/L] :- myo-inositol [ 100 ], thiamine [10 ] , nicotinic acid [1], pyridoxine [1], folic acid [1] , glutamine [100], glycine [10], ....................................proline[10]

Recipe - make up three solutions as fractions for total of 1 litre;

Part A KNO3 - 620 mg, Ca(NO3)2 - 320 mg, Mg(NO3)2.6H2O - 70 mg, protein hydrolysate - 4g, sucrose 30g, agar - 7g, activated charcoal - 0.8g
Part B KH2PO4 - 120 mg, KCl - 10 mg, H3BO3 - 1.8 mg, Na2MoO4.2H2O - 0.1mg
Part C MgSO4.7H2O - 190 mg, 100x cationic micros - 10 ml , 100x vitamins - 10 mls, K-OA* - 6.7 mls, NH4-OA* - 4.85 mls. (* = see below)
(the protein hydrosylates I use are lactalbumin hydrolysate and soya-peptone 2g of each; also the MgNO3 could probably be omitted as there is enough from the MgSO4)

Part A is in the larger volume, Part B and C in smaller volumes adding up to 1000 mls total. All are adjusted to pH 5.0 (final pH of medium 5.0 - 5.2)
Part A is heated to dissolve or part dissolve the agar then Part B is added with stirring. This is then autoclaved.
Part C is best in a volume that can be filter sterilized into a sterile bottle. This is then heated to about 55-60C in a water bath while the autoclave is cooling.
Part C is added with stirring to the combined Part A+B under sterile conditions when the temperature of the autoclaved medium has fallen to below 65C.

I make up 350 mls total at a time, large volumes are not practical for me. So part A is about 250 mls, part B is 50mls and part C is 50 mls. I use a 500 ml conical flask to make up part A, B is made up in a small beaker and preheated before adding to A while swirling the flask to mix during agar dissolving. In the glove box the filter sterilised and preheated part C is added with swirling for at least 60 seconds to mix it in well. This is allowed to cool to approx 45-50C before pouring out into pre-sterilized polypropylene deli containers.

I make up stock solutions for most of this. So part A salts are kept as a 100x stock with about 0.1 mM malic acid and adjusted to pH 4.0 and with 0.1 % benzoic acid added as a preservative, it keeps at room temp in the dark for over 6 months without contamination. The same for salts in part B.

Part C is a bit more complicated. The MgSO4 is made as a small stock each time I make a batch of media. The chelated cationic micros, the vitamins, the K-OA and NH4-OA are all stock solutions which are aliquoted and frozen.

The Fe is chelated with EDTA [ 54 ], the other cationic micros are chelated with a mix of glutamate, aspartate, histidine and proline,
each at about 0.08 mM (final, i.e. medium concentration, stock solution is 100x this). The chelation is done by combining solutions heated to about 70C prior to mixing metal ion and chelator solutions (adjusted to pH 5.0). The Fe-EDTA and the other chelates are combined to make 100X stock and stored frozen (-20C) as aliquots. Basically four separate solutions, (EDTA + Fe) and (Mn,Zn,Cu,Ni + amino acids) are all combined so as to give a 100X stock, so starting concs are 400x.
I haven't given the recipe figures for the cationic micros as there too many versions to choose from, but if anyone wants the figures I can dig them out. I guess that chelation is not necessary and any standard medium micro mix could be substituted.

The K-OA and NH4-OA are the complicated stocks to make. I add 1.5 g each of the 4 organic acids to about 60 mls of water, then I add NH4OH or KOH at 2.5 M from a burette and slowly bring the pH up to 5.0 with stirring. I record the final volume of alkali added, to calculate the weight fraction of K and NH4, and the combined weight per ml in the final stock when made up to 100mls. Because concentrated NH4OH is so volatile there is some error involved, and the KOH titration helps to gauge any loss of NH4 on storage because the molar fractions at the target pH are similar when using new unopened NH4OH stock (25% volumetric [18 M], although a 15 or 20 % volumetric preparation of NH4OH would be preferable).

I'm thinking of leaving out the oxoglutaric acid (alpha keto-glutaric acid) as it's unstable and I don't have room to keep it in the fridge. In terms of buffering it's pKa's are both on the acidic side of the useful range. Malic acid has a pKa at 5.1, succinic acid at 5.6, and citric acid at 4.7 and 5.8 (this higher pKa for citrate is found in text books to be 6.4, but recent determinations at the low millimolar range concur that it is around 5.8 at these concs). Succinic and oxoglutaric acids have been found to be more effective at reducing NH4 toxicity than either malic or citric acids ... the reasons for this have been debated, but IMO not resolved. In formulation I try to keep the total concentration of organic acids a little higher than the NH4 concentration, which is why I also add the K-OA.

I'm sure a lot of this complication is not necessary, and hopefully I will get around to simplifying this in time, but as it works very well, my main focus will be on the replate medium.

 

[myxodex]

The replate medium

I will give details in subsequent posts, as it isn't yet clear that the most promising medium is successful, time will tell.
This is the story of how I ended up making the most promising medium (I actually label it as SWM).

The first replate medium I made up had reduced NH4, but I couldn't bring myself to add the 60g/L sucrose as in Chung's recipe, I reduced this to 40g. It didn't work. Second attempt I added 60g. It didn't work, but the seedlings died off a bit slower. It could be that the sugar I'm using: pure cane sugar - supermarket grade - is the problem. It worked OK for the germination medium. It is possible that this grade of sucrose is toxic at 60g, but not at 30g and without lab grade sucrose I cannot answer this problem. Also because I cannot read the details in Chung's paper I don't know whether he filter sterilised it or autoclaved it.

So I did some research on osmolarity in tissue culture media. I came across some papers on rice propagation and they solved their problems by using maltose instead of sucrose. Maltose turned out to be quite interesting. Generally it isn't as efficient as sucrose is as an energy source in tissue culture; one paper described it as a "slow sugar". It did seem that with some species it was good at promoting development, reducing stress, and the browning of media from phenol oxidases in some woody plants. One paper had a model that excessive sucrose results in the build up of high levels of hexoses in the cytoplasm and that this represses protein synthesis rates. It also turns out that maltose is the main sugar released from chloroplasts at night from starch breakdown in chloroplasts. Sucrose at high concentration worked for Chung, but I decided to try using 30g sucrose + 30g maltose as the osmolyte.

The problem was where do I get maltose. I tried maltose syrup from a Chinese supermarket but this didn't work, the syrup was yellow brown and it is known that compounds such as hydroxymethylfurfural (and related compounds) produced from prolonged heating of sugars is toxic for plants in tissue culture. Then I came across the fact that sweet potatoes have a high concentration of beta-amylase; this degrades starch to maltose. I found a highly soluble form of starch sold by sports supplement vendors called Vitargo. I made a smoothie from sweet potato in a blender, filtered out as much of the solids as I could and used this in a 6 hour digest of a concentrated Vitargo starch solution (malic acid buffer pH 5.0 @ 50C) it went from tasteless to sweet. This is what I used in the most promising replate medium, or SWM. So 30g sucrose + "30g equivalent" of the starch digest. At the same time I tried another osmolyte; iso-malto-oligosaccharides or IMO, also sold by supplement vendors, this one didn't work but the seedlings seemed to be surviving, just not growing.

What I don't know is whether the sweet potato extract - starch digest has hormones or other nutrients that my medium lacks or whether it's the maltose in this. More recently I got an email from the new sales rep at the same scientific supplier that agreed to sell me the organic acids,.. and yes, they could sell me some USP grade maltose. So the next experiment is pure maltose vs sweet potato starch digest ... I really hope the pure maltose works, it would make life so much easier, ... but I have a sneaky feeling that I'm going to be making more sweet potato smoothies and 6 hour digests. The worst bit is that I don't have a temperature controlled water bath, I use a slow cooker on its lowest setting and it slowly climbs up to more than 60C, so I have to watch the temperature and switch it off and on periodically to keep it at 50C. Damn ... I really need a small laboratory !