Green Pod Sowing

This is basically the second half of an earlier post in which I flasked up some Epidendrum seeds.  I finally (after the intervening holiday) got around to sowing the green pod.

Green Seed Pod of an Epidendrum Orchid
©2011 MadOrchidist.com

The seeds come from the same "Pod Parent" (mother plant) as the dry ones, but there is no way to know if the same pollen got into both flowers, or if the two pods have different "Pollen Parents."  I also don't know when they were pollinated, so I cannot be sure that the seed in this one is mature enough to germinate - only one way to find out . . . 

The procedure for this one is a bit more straightforward than for loose seed because the capsule is sealed.  Theoretically, the seeds inside have had no contact with the outside world and thus are uncontaminated by bacterial and/or fungal spores.  That's one of the things that draws me to this method, so I hope it works out.  Since I have sterile mother flasks all made up, the main points are: sterilize the outside of the pod, cut it open, and get the seeds into the flasks without getting anything contaminated in there.

Toothbrushes are so versatile!
©2011 MadOrchidist.com

To begin sterilizing the outside of the seed capsule, I first soaked it in a 1:4 bleach solution with a tiny bit of soap surfactant.  I shook up the jar and brushed at the pod a bit with the toothbrush periodically while I autoclaved some distilled water (for rinsing later) along with my scalpels and forceps.

That probably would be good enough, but I have also seen green pods soaked in alcohol, and even lit on fire!  That sounded right up my Mad Scientist alley, so I gave it a shot:

Here is a quick video I shot outside the glovebox (with one hand, while working with the other), of flaming the green seed pod.  It is probably overkill in this case, but it looked fun and I wanted to try it!  I don't think I'll make it part of my regular procedure because I don't want to melt or blow up my sealed plastic box, and there isn't much benefit to doing it outside of the box (except the fun factor, of course).

Then it was back into the bleach and into the glovebox with my tools.  Once the box was sealed and clean, I scrubbed at the capsule a bit more and then rinsed it in the sterile distilled water.

I use a small plastic cutting board in the glovebox as a work surface.  Covering it with aluminum foil makes a quick and easy to sterilize cutting surface with the added benefit that cleanup is easy.

Ready to cut open the seed pod
©2011 MadOrchidist.com

Then, the big moment; I cut it open.  Here is what the capsule looks like inside:

Orchid Seeds!
©2011 MadOrchidist.com

The above photo is of the leftovers, after I was done.  There was more of the same whitish seed and fibrous material initially, and some of it is now in flasks:

Seeds In Vitro
©2011 MadOrchidist.com

I Just scraped some out with the edge of the scalpel and tapped it in.  Once the flasks were sealed again, I tapped and shook them around a bit to distribute the seeds across the surface of the nutrient media.

I opted to make up three flasks, as with the dry pod.  I will be anxiously awaiting any comparisons I can make between the two groups.  If these do not germinate, the pod may have been too immature, but as long as no contamination grows, I will consider it a success either way!

Flasking My First Orchid Seeds

This is what it's all about; sowing some Orchid seeds in flasks!  A couple of my own crosses capsules' are nearing maturity, but I couldn't pass up the opportunity to practice on some that just came along.

Epidendrum seed capsules ready for flasking
©2011 MadOrchidist.com

The other night was the Orchid Society Holiday Party.  Our host had some Reed-stem Epidendrums out in the yard that he says set seed all the time.  As it happened there was a dry pod that had split open and a green one still sealed, both on the same plant.  I have been considering whether I should go with the "green pod" method or wait for my pods to spit naturally and then sterilize the seed, and here was an opportunity to try both and compare!  He let me take them both to sacrifice to the learning process in my lab (thanks Van!).

Pod Parent Tag
©2011 MadOrchidist.com

Given that the Epidendrum was probably pollinated by an insect, I have no way of knowing what the pollen parent was.  All I know for sure is that the pod parent is pink; its tag is pictured above, plus I saw a few of the flowers on another spike.  Since this is sort of an experiment, I am not really worried about the pedigree.  If I end up growing anything from these they will be good for landscaping; Reed-stem Epis are practically bulletproof outdoors in this climate.  Let's hope the seeds are hardy too:

Epidendrum Orchid seed capsule dried and split open (dehisced), fine seeds visible
©2011 MadOrchidist.com

I decided to deal with the open capsule first.  Once an orchid seed pod dries, it splits open to release the thousands upon thousands (sometimes even millions) of tiny seeds inside.  After this happens, the seeds must be considered contaminated and their surfaces sterilized before sowing.

Epidendrum Orchid Seeds being sterilized in a syringe
©2011 MadOrchidist.com

I opted to use household bleach in a ratio of one part bleach to nine parts distilled water as my sterilizing solution.  Several online tutorials and discussions recommended using a syringe to clean the seeds.  I bought dispensing syringes with caps on Amazon.com and made one cap into a filter by melting a couple of holes in it with a heated pin and then placing a small piece of coffee filter inside.

Basically, I drew up about 10mL of the sterilizing solution through the filter cap and then banged the syringe on my hand and the table for at least ten minutes to try and eliminate all of the tiny air pockets that cause the seeds to float and prevent the solution from doing its job.  Below is a close up photo of the tiny orchid seeds in the syringe of sterilizing bleach solution:

Tiny Epidendrum Orchid seeds floating in sterile solution
©2011 MadOrchidist.com

 I put the syringe, several jars of autoclave sterilized distilled water (for rinsing seeds), a few pre-made flasks with seed sowing media, and a jar of stronger bleach solution (to clean the outside of the syringe and any tools I used) into my Glove Box and sealed it up.

Tools and materials in the glove box
©2011 MadOrchidist.com

It is unfortunately impossible for me to have my hands in the gloves working and take pictures at the same time.  At some point a small tripod and/or an intrepid volunteer will enable me to post more photos of what goes on inside the sterile glove box.

Briefly, I squirted out the bleach solution, drew up a rinse of distilled water a couple of times, and then squirted a small remaining amount of water with the seeds into a few flasks.  The result is pictured below:

Tiny Orchid Seeds in Flask
©2011 MadOrchidist.com

I got some seeds on the sides of the flask, but there are plenty in there on the agar media too.  I also ended up with more water than I would have liked in the flasks, so we'll see if that hurts things any.

I'll try to get to the green pod tonight or tomorrow so that I can make a fair comparison of the results.  I also received some seeds today in the mail that I ordered from Thailand on eBay.  I think I will give today's flasks a few days to see if contamination develops, so I can change my methods if necessary before trying again.  For now, those seeds will go into my pollen storage unit in the refrigerator.

As for these flasks, now we wait . . . it's a theme with orchids . . . 

Failure is Always an Option

Well the point of this blog is, at least partly, to keep track of my Orchid culture progress, so I would be remiss not to include the occasional failure.  Here are a few things that have not gone according to plan lately.

These first few photos are of flowers that were pollinated, but then withered away without developing a seed pod.  I am willing to give up enjoyment of the flower in return for a seed pod, but when you lose both it really stinks!

Cattleya flowers that were pollinated, but never went anywhere
©2011 MadOrchidist.com

Another Orchid Dud!
©2011 MadOrchidist.com

Laelia anceps that never developed pods - bummer, I had high hopes for these . . .
©2011 MadOrchidist.com 

These next few pictures are of the Dendrobium nodes that I flasked up in this post.  All three of them developed contamination.

contaminated Dendrobium node flask
©2011 MadOrchidist.com

The same flask as above, a few days earlier - that stuff grows fast!
©2011 MadOrchidist.com

The other two flasks developed fuzzy fungus
©2011 MadOrchidist.com

I had acknowledged previously that stems are tough to get completely sterile for this type of culture; now I know first-hand.  I could try to clean and replate them, but they were really just for practice and I'll probably let them go.

So, with some failures under my belt, I feel like I am making progress.  Next, on to some seed sowing . . .

Orchid Pollen Storage

Since I have been pollinating my orchids, I have obviously been harvesting pollen.  In addition to using it fresh to cross two blooming plants, I have been saving some pollinia in the refrigerator for future use.

Supposedly orchid pollen can stay viable in the refrigerator for months, allowing crosses between plants whose blooming times don't coincide.  I already have at least one seed pod developing nicely that was made with refrigerated pollen.

Well as I began to make more crosses, I accumulated more and more folded paper pollen packets in my refrigerator.  Today I decided to upgrade the way they were stored and organized.

Orchid Pollen Storage in the Refrigerator

I used a small 15 drawer organizer that previously held random screws and bits in the garage.  Now, every time I open the refrigerator I smile at how geeky this looks . . .

Sibling Rivalry

This post is about one of the things that is attracting me to Orchid Breeding: genetic variability.  I have two different specimens of a particular orchid cross blooming right now, and they are an excellent example of the variation that is common.

2 Cattleya Claesiana x Laelia milleri siblings
©2011 MadOrchidist.com

These two plants are siblings from the same cross - Cattleya Claesiana x Laelia milleri.  Both plants are originally from the Santa Barbara Orchid Estate (though I got at least one of them secondhand) and have been grown under similar conditions.  Their site features another flower from this same cross that seems somewhere between my two:

Photo from www.sborchid.com

The blooms vary in most of the parameters one would want to look at: flower count, size, color, orientation, etc.  Even the plants themselves are quite different in size.

Cattleya Claesiana x Laelia milleri
©2011 MadOrchidist.com

The one on the left has a single flower, and it "looks up" in a way that is typical of many of the small rupiculous (rock growing) Laelias, the group to which milleri belongs.  However, milleri has multiple flowers per spike, like the plant on the right.  Those blooms are a salmon-ish pink-ish orange-ish color compared to the cooler pale magenta of the single flower - a color that is probably from the Claesiana parent.

Here they are together in one photo for direct flower size and shape comparison:

Cattleya Claesiana x Laelia milleri
©2011 MadOrchidist.com

I really enjoy the mental exercise of trying to figure out which parent contributed what quality, though it is impossible to know for sure.  You can see, just from these two, that when carefully selecting parents to make a cross you won't always end up with something in between.  You could end up with the best or worst qualities of both, or an interesting shuffle as with these . . .

Laboratory Upgrade!

Today my Home Laboratory got a big upgrade!

Laboratory Stainless Steel Table!
©2011 MadOrchidist.com

 My boss gave me an old metal restaurant/laboratory table that the business no longer had a place for (thanks Pat). It has an 8 foot long, 2-1/2 foot wide stainless steel surface; you should have seen me unloading it from the truck by myself!  It has plenty of space to move all of my equipment out of the house and set up in the garage.

Moving the Equipment In
©2011 MadOrchidist.com

Once I moved everything to its new place, I got my dining room table and kitchen back!  Here is a picture of the situation before:

And After:

Now That's Sceincey!  Note My Antique Dissecting Scope At The Left.
©2011 MadOrchidist.com

There is even room for my insect collection stuff at the other end of the table.  Now all of my Mad Scientist fun can happen in one dedicated place . . .

Node/Keiki Culture - Dendrobium Lifeboat

I have mentioned previously that my Grandfather was an Orchidist, and even though he passed away years ago, my Grandmother still keeps a few orchids around the house.  These days they are mostly store-bought Phalaenopsis in the windowsill and a few random things out on the patio.

When I went to visit Grandma the other day, I noticed some dry Dendrobium canes leaning in an empty pot outside.  She is nearly 90, so she doesn't make it out there to water very often, and between that and the recent cold, this plant had wasted away.  The roots were dry and cracked (not even potted in any mix), and there wasn't a leaf to be found.  There were even a few well developed keikis that had made a start, withered, and died.  Even so, I noticed a few nodes that were developing a last ditch effort at new plants.

Dendrobium lifeboat!
©2011 MadOrchidist.com

I was offered the plant if I thought I could save it.  No way, but I realized these developing nodes would make a good experiment in my new mad laboratory.  I knew Phalaenopsis could be cultured in vitro from nodes on flower spikes and figured the same would be true of these nascent keikis, especially since they had already started to develop.  There's only one way to find out!

Cane Nodes cut from a dead(ish) Dendrobium
©2011 MadOrchidist.com

I brought it home and cut the canes up, leaving just a bit on either side of the three nodes with developing keikis I thought looked viable.  I realize now that I should have made more effort at this point to get all of the dry papery covering off, as this may be a source of contamination later - we'll see.

Orchid cuttings in sterile solution
©2011 MadOrchidist.com

I soaked them in a mild bleach solution for about 10 minutes, and prepared some sterile distilled water while I waited.  I already had empty flasks made up with media, and I didn't want to run the pressure cooker autoclave for just one jar of water, so i microwaved some distilled water in a jar.  I actually set the lid on too, which I know is not usually a good idea in the microwave, but its smooth surface did not allow any sparks to occur.  While not ideal, I hope this water is sterile enough to rinse my cuttings without adding contamination.

Boiled Distilled Water
©2011 MadOrchidist.com

Once the water cooled, I placed it, the cuttings (in sterilizing solution), and the necessary implements into my glove box, closed it up, and sprayed everything with bleach.

Tools in the Glove Box
©2011 MadOrchidist.com

Below is my view through the clear acrylic window in the top of the Glove Box:

My View while Working in the Box
©2011 MadOrchidist.com

Basically, I just scrubbed the cuttings with a toothbrush, cut off a little more at each end, and then placed each one into its own baby-food-jar flask.

Flasks in the Glove Box
©2011 MadOrchidist.com

Here is the result:

Developing Dendrobium in Flask
©2011 MadOrchidist.com

Result: More Orchids!
©2011 MadOrchidist.com

 There was actually one small "new lead" developing at the base of the withered mother plant too.  It had new root tips developing, and I opted to pot it up in sphagnum moss and try to grow it alongside the node flasks for comparison.

I have read that other folks doing this kind of culture at home have had a hard time with contamination due to the difficulty of completely sterilizing the cuttings.  Only time will tell if these proto-orchids will grow in the flasks, or something gross will grow instead!

Frickin' Freezin'

Well, after boldly proclaiming in my last post that Laelia anceps will hardily survive frost events, I woke up to ice on my windshield!

Not Orchid Weather!
©2011 MadOrchidist.com

I haven't bee out to the Orchid Zone yet to do damage control, but I hope that being up against the house has spared the more sensitive among my collection.

Here is an update on my recent Dendrobium pollination:

Dendrobium Hamana Smile seed pod growing
©2011 MadOrchidist.com

After just one week, the ovary is already very swollen, and a clear seed pod is developing.  I think that these Australian Dendrobiums have very short pod development times compared to other popular orchids.

I have kept this one on my desk since bringing it inside, and the fragrance is just wonderful!  It now has at least 15 flowers open on 4 spikes, and I have selfed a few more of them . . .

Anceps and Updates

December is definitely Laelia anceps season.  This hardy orchid from Mexico blooms in the winter, despite the coldest temperatures of the year, and will routinely survive frost events.

In addition to all of the wonderful hybrids this species has parented, there are innumerable varieties of the pure species itself.  I probably have six or seven different kinds and often feel the "collect the whole set" urge to get more.  In fact, I am toying with the idea of mounting them all on one large cork tube to make a Laelia anceps tree; it would be quite a sight for the holidays!  I am fortunate to live near one of the worlds premier anceps growers and hybridizers, The Santa Barbara Orchid Estate.  Their Laelia collection is second-to-none, and most of my specimens of that genus have come from there, whether directly or indirectly.

So, here I am looking for blooming orchids to pollinate in December, and it isn't surprising that these beauties are on the list:

Laelia anceps var. 'Veitchiana' x 'Irwin's'
©2011 MadOrchidist.com

The one on the left is the variety 'Veitchiana' and is the pod parent in this cross.  On the right is the 'Irwin's' that I used in a previous cross; it's flowers have already faded after being pollinated previously (pictured below), but I kept some of its pollinia in the refrigerator.  I also placed the 'Veitchiana' pollen in folded up paper and added it to my pollen "cold storage" in the refrigerator.

Laelia anceps pollinated earlier; note the slight swelling of the ovary
©2011 MadOrchidist.com

A couple of my earliest attempts have yielded nice fat seed pods, and at least one is looking very ripe.  I am still deciding if I want to use "green pod" seed sowing methods, or wait for dehiscence (the natural splitting open of the pod) and sterilize the seeds afterward.  This pod may make that decision for me by bursting!  Given that it has begun to turn yellow, it is probably ready for "green pod" sowing now, but I want to give my empty flasks at least a full week or more to be sure they are not contaminated.

Maturing orchid seed pod at 16 weeks, approaching dehiscence
©2011 MadOrchidist.com

Speaking of contamination, the "contamination control" flask from my last post still looks clean, let alone the ones that are supposed to be clean.  However, I did make up some Petri Dishes with the excess agar media mixture, and those are growing some interesting (or perhaps unsettling, depending on your viewpoint) things.  The first one I left exposed to the air, just like my "contamination control" flask, but for longer.

Airborne contamination growing in a Petri dish
©2011 MadOrchidist.com

The second one was wiped with a small spatula that my Scottish Terrier, Dr. Watson, had licked.  Not surprisingly, his mouth is not sterile (even though he has been sterilized)!

Dog-borne contamination!
©2011 MadOrchidist.com

Both plates show representatives of the two major groups of contamination: bacteria and fungus.  The fungus initially shows up as fine hyphae (threadlike structures) and eventually gets "fuzzy", while the bacterial colonies tend to look "slimy."

Hopefully none of this will show up in my batch of baby-food-jar flasks, and I'll be sowing seeds before the end of the month!

UPDATE:

Here is a photo of the same Laelia anceps 'Veitchiana', 24 hours after pollination.  You can clearly see that the petals are folding forward over the column.  To speak teleologically for a moment, the plant doesn't "want" pollinators visiting a flower that is already pollinated, so it immediately makes it less attractive.  Flowers are "expensive" to maintain (especially in terms of water loss), so the sooner one gets pollinated and then discarded, the better from an evolutionary biology standpoint.

Laelia anceps after pollination
©2011 MadOrchidist.com

However, I can see why most orchid hobbyists are reluctant to pollinate a beautiful flower that they want to enjoy for a while . . .

Under Pressure - preparing flasks

I am continuing to collect equipment and supplies for my Orchid Flasking adventure and have taken another couple of steps closer to success.  A key component of that success will be sterility, and a key tool for that is an autoclave.

Mine was actually designed as a home pressure canner for preserving food (a giant pressure cooker), but that process has the same end in mind: a final product completely free of bacterial and fungal contamination.  As long as it can sustain at least 15 psi of pressure and 250° F it will accomplish that.  In my mad-scientist way, I rather enjoy the anthropomorphic thought of contaminating organisms landing in the freshly made nutrient media and "thinking" they have hit the jackpot, only to be utterly destroyed by this vintage piece of kitchenware.

The Beast - My Giant Pressure Cooker Canner
©2011 ColinFraser

Speaking of vintage, this thing is a classic.  It is a huge 20-Quart Presto from the 1970s and doesn't have any of the annoying safety interlocks of its modern counterparts.  The "Harvest Gold" enamel color attests to its age!  I bought it on eBay for $31 (plus some serious shipping - it's heavy), and I think I got a great deal because the "Harvest Gold" color has fallen out of style.  It reminds me of a Gibson Les Paul guitar from the 1950s whose mustardy color came to be known as "TV Yellow."  It was designed to show up well on black-and-white TV, but wasn't so popular after color caught on.

So, what goes in this monster?  Well, I ordered some pre-measured P668 Orchid Seed Germinating Media from the Orchid Seedbank Project.  Aaron sells it in packets that you just add to a liter of water and dissolve - simple.  I have also been collecting jars to put the media in.  My boss has been kind enough to set aside empty "palm hearts" jars for me, and I think those will make excellent replate flasks, but the most popular thing for Amateur Orchid Seed Germination ("mother flasks") is probably the 4oz baby food jar.  I don't, and never have had, kids; how else would I have time for all this Orchid Madness?!  So, I went back to eBay looking for empty jars.

Cleaning Jars in the Sink
©2011 ColinFraser

I ended up ordering some, and with shipping it worked out to a little over $0.50 per jar (the cheapest I could find them).  During the days while I waited for them to arrive in the mail, I actually looked at baby food in stores - the first time since I had to buy pureed fruit for my breeding Day Geckos years ago.  Imagine my surprise when I discovered that Walmart (I know, I know) has cheap baby food for $0.50 per jar; the food inside is worth less than the glass jar is!!  I could buy full jars, not wait for them to be shipped, feed the contents to my Scottish Terrier, and have them ready to go for the same price and less time than ordering them empty online!  What a crazy world . . .

Preparing to Fill the Clean Jars/Flasks
©2011 ColinFraser

I bought and emptied a few more jars, and my eBay purchases eventually arrived.  I removed the labels and any remaining glue, rinsed them well, soaked the jars and lids in hot bleach water in the sink for a while, and then rinsed them again.  Now my jars were ready to become flasks.

I added the media to a liter of distilled water, brought the mixture to a boil on the stove, and stirred while everything dissolved.  I let it cool slightly and then dispensed it into the jars with a "flavor injector" syringe.  There are almost 34 ounces in a liter, so it was no problem filling 30 jars with one ounce each.  I then put the lids on LOOSELY and arranged them all in the pressure cooker.

Following the manufacturers instructions (which I found online), I sealed it up and then vented the chamber for 10 minutes.  Once steam had displaced all of the air inside, I placed the pressure regulator over the vent, and the pressure began to build.

Sterilizing Heat and Pressure
©2011 ColinFraser

 When the gauge reached at least 15 psi and the regulator began to "jiggle,"  I let it run for at least 15 minutes at that level, before turning off the heat and letting it cool slowly.  My media was now autoclave sterilized and my "jars" had been transformed into "flasks."

The Agar in the Media Turns to Gel
©2011 ColinFraser

As the flasks cooled the media solidified, and I tightened the lids as I removed them from the pressure cooker.  I also intentionally opened one flask and exposed the media to the air for a few moments.  This will serve as my "contamination control" flask; it is virtually guaranteed to be contaminated.  When growth of contaminants is detectable in this flask, I can check it against the other ones, which will still hopefully be pristine.  At that point, I know that the remaining flasks are safe to use for Orchid Seed Sowing.  Neat huh?

Clean and Sealed
©2011 ColinFraser

Finally, I placed the cooled and sealed flasks into my still unfinished glove-box and misted bleach solution on and around them.  I let them sit for a bit; then I donned a pair of gloves, wiped each one, and wrapped some cling film around the lids.  Hopefully, this will prevent the entrance of contaminants if the flasks "breathe" a bit with changes in temperature and pressure.

Now all that's left to do is wait a couple of weeks and see how much, if any, contamination develops.  I have a couple of seed pods that are nearing readiness, so these flasks should be ready just in time!