Saturday, December 29, 2012

Fiery start to the 2013 orchid season

Voëlklip, Fernkloof Nature Reserve begins to burn
Flames march down towards the houses
As I report this, several Huey fire-fighting helicopters and a large Oryx from the South African National Defence Force are flying low repeatedly over our house in Voëlklip in Hermanus collecting seawater from the beach just streets away and bombing a large wild fire that is raging in the Fernkloof Nature Reserve and threatening homes just streets above us towards the mountain. The fire is being fanned by strong North-westerly winds and the road (R43) from Hermanus to Stanford has been closed. The fire is travelling fast and came over the top of the mountain from the Caledon side two days ago. It then moved West from Stanford towards Hermanus. It arrived opposite us at 03:20 this morning. Since I took the photos of the mountain early this morning the fire has moved down the slope towards the homes. The fire-fighters and other emergency service personnel are doing an amazing job and I take my hat off to those pilots, guided by a spotter plane who are continually bombing the fire line. I managed to take a few pics of the Oryx which had to land this morning to wait for a re-fuel. Ironically, I found a dried out Disa bracteata just a stones throw away from it!
Oryx waiting for re-fuel

Water bombing bucket
On the upside, the fire comes at a good time for the many orchid species in Fernkloof Nature Reserve. I counted 69 listed terrestrial orchid species on the list of plants from Fernkloof Nature Reserve. Most have probably already set seeds and have dried out in time for their Summer dormancy. Many of the South African terrestrial species actually need periodic burns to thrive which forms a critical process for the Fynbos of the Cape Floristic Region. The Spring season of 2013 will be especially rich with orchids and other plants which will be out in numbers after this fire. I am looking forward to this. A good webpage that describes the relationship between the local plants and fires can be found by following the link to the Fernkloof Nature Reserve webpage: . I guess one thing we residents will need to keep an eye out for now is the exodus of wildlife including venomous snakes coming down from the mountains to escape the flames. We already have some interesting birds in the garden which we have never had at home before. We have several Cape Sugarbirds (Promerops cafer) taking up temporary home in our large bottlebrush tree.

Friday, December 28, 2012

Some tissue culture tips and success secrets

I was browsing through the stats on the blog recently to see what subject seems to be the most popular. It appears as though there are a lot of would-be amateur tissue culture hobbiests out there with a keen interest in how to get started but also how to get things done properly with little fuss. I have read some interesting articles on the internet about home tissue culture of orchids and what methods to follow. Most are basic repetition of the blah-blah basics with some useless stuff perpetuated. Some are good though, especially those which dispense with the drivel about useless or over-complicated techniques. One such "technique" is the tea-bag or waterproof paper technique for surface sterilising dry orchid seeds. Equally irritating is the syringe-method which I threw out many moons ago (in fact about a week after I started). So... since I am abviously irritated by things that challenge my impatience I have decided to share some simple techniques with you that actualy work. In fact, so much so that my entire seedling collection is based upon these since most were cultured as dry seeds.  

Dettol hand sanitiser
In previous and slightly aged posts, I indicated the importance of understanding the concept of sterility. This is a crucially important first step. Sterility does not mean clean or disinfected, it means absolutely sterile - ie - nothing living! Now consider that everything that is not sterile is covered in bacteria and fungal spores and other microscopic organisms that are just waiting for an opportunity to spread and to multiply. Your basic view now should be that of an obsessive compulsive with a phobia of germs! Possible transmission routes include YOU (your skin, hair, breath, clothes etc.), the air, all surfaces, your tools and kit and the orchid seeds themselves. Some of the most stubborn of bacteria I find are those associated with the actual seeds! Ok, so most of you would be aware that absolutely everything would need to be sterilised before success can be achieved with germinating orchid seeds in vitro. My archived post on building and using my laminar flow hood should provide you with some basic and useful information on building your own hood for orchid seed use. Although you can do seeds in a glove box or sterile still-air cabinet which I also did at the very beginning, you will never achieve the rate of success that you will with a laminar flow hood. It's worth spending the time and the bucks to make it. Mine cost approximately R2000 for all the materials and construction. For the US readers this equates to about $250. All my tools are cleaned before being sterilised in a 10 quart 15/16 bar pressure cooker for 30 minutes. I wrap all my tools in aluminium foil first so that I can remove them without actually touching them and can transfer them to the laminar flow hood where they can be carefully unwrapped under sterile air and placed into a sterile jar ready for use. I also try not to store too much ready-made plates of media in the fridge and prefer to make a batch of new media the same day or the day before and to allow these to set in the laminar flow with the Hepa running until I use them (so plan ahead - it helps avoid unnecesary non-sterile exposure). Wash your hands and forearms well before working in the hood. Use a good non-sticky hand sanitiser like the one made by Dettol (kills 99.9% germs). Use this liberally on your hands, making sure you get it under your nails and remember to remove your rings and watch before working. I routinely wipe down the inside of the hood working chamber with neat sodium hypochlorite solution before commencing with any work. When the hood is not in use I have a hinged door that seals off the front of the chamber to prevent incidental aerial contamination that might threaten the viability of my Hepa. 

Holstered auto-pipette and pipette box
Now for the good stuff. Buy yourself an automatic pipette like the one in the photo. These often come in standard starter packs supplied by almost all of the lab consumable companies. They are a standard feature of molecular laboratories. However, ensure that the one which you get is fully autoclavable. The unit can be wrapped in foil and autoclaved as for all the other tools. Those which are not fully autoclavable can be used but you will need to familiarise yourself with all the internal parts and how to dismantle and reassemble them. All these parts must be placed into a plastic container and completely covered with a 40% hydrogen peroxide solution for 30 minutes before rinsing in sterile water and reassembled ready for use (so... just get those which are fully autoclavable - it is MUCH easier). The starter packs usually also come with pipette tips supplied in an autoclavable box. If not, just get a box of tips and a bag of replacement tips from the same supplier to fit your pipette. These are dead-cheap and disposable and fully autoclavable. I suggest that you get yourself either a 200µl or 1000µl pipette and tips. These two sizes are often the most common and are also the most appropriate for the job depending on the working space which you have. I use 200µl pipette and tips.

Now purchase a bag of eppendorf tubes from the same lab consumables supplier (see pic). There are various volumes to choose from but get the graduated 1500µl or 2000µl tubes with lockable caps. Your next investment will be a vortex mixer (see pic) which will ensure that the seeds will be continuously aggitated and mixed with the sterilising agent for the correct amount of time and will avoid dead-spots associated with other techniques. Vortexing in the eppendorfs also concentrates the seeds nicely allowing better control for rinsing and pouring of seeds onto plates. Eppendorfs are also autoclavable and I place a fistful into a polypropylene tub with a sealable lid dry into the pressure cooker for sterilising. Once sterile, I remove this tub of eppendorfs to the laminar flow. You can include the vortex mixer inside the laminar flow if you have the space but you can also have it next to the hood on the working bench if you wish. In the hood you should also have a sterile flask of RO water and a flask of pre-made sodium hypochlorite solution to which some surfactant has been added. Use the following concentration of non-perfumed liquid sodium hypochlorite: 120ml tap water to 20ml neat household sodium hypochlorite solution (3.5% m/v). Add a few drops of Tween20 or household dish-washing liquid (not too much or it will foam too much!). The surfactant facilitates good soaking by allowing the sodium hypochlorite solution to "stick" to the surface of the seeds.

Vortex mixer
Sterile eppendorfs
When ready to start, put the dry seeds onto a folded piece of paper and funnel into a sterile eppendorf tube. Inside the hood under full flow, push the pipette onto a tip in the box and close the box again. Do not allow the pipette or tip to touch any surfaces unintentionally while working. If this happens discard the tip by depressing the eject button on the pipette and begin again with a new tip. Take up a metered solution of sodium hypochlorite (200µl if you are using a 200µl pipette and tip; the pipette is adjustable to volume - pre-set the volume before you sterilise) and add repeatedly to fill the eppendorf to about 3/4 full. Holster the pipette and close and lock the eppendorf cap. Vortex on high for 5 minutes. Shake initially with your hand before vortexing to loosen any seed clumps. At 4 minutes remove from the vortex mixer temporarily to view the buoyancy of your seeds. If they are nearly neutrally buoyant/slighly heavier than the solution, allow the final minute for the seeds to settle at the bottom of the eppendorf tube. If they sink quickly, you can allow them to settle at the end of the 5 minutes. Some seeds with float, especially those of terrestrial orchids. Allow these to collect at the miniscus. For seeds that collected at the bottom of the eppendorf carefully pour off the sodium hypochlorite solution inside the laminar flow hood into an awaiting empty waste receptacle which was also previously sterilised in the pressure cooker. The seeds will remain at the bottom of the tube after the solution has been poured off. Hold in one hand inside the hood and with the other hand charge the pipette with a new tip (expel the old tips into the waste receptacle) and take up sterile RO water and add to the tube to 3/4 full again. Close and lock the cap and vortex again for 30 seconds. The seeds will appear heavier in the RO water (sinking faster) because of the reduced specific gravity of the solution. Allow seeds to collect again at the bottom of the tube and pour off all the water. Repeat again with a new tip and RO water and vortex to mix all the seeds again. This time, pour the entire contents onto your awaiting plate and seal. Floating seeds are a bit tricky because you have to work them to the bottom of the tube by holding the tube horizontally while carefully twisting it from side to side between your fingers. You will lose some seeds during the above process but with experience you will get a feel for it and get to a point where you lose almost nothing.

I do all of my dry seed work as above. I have found it produces superior results and minimises contamination. I hope you found this post useful. I would love to hear your thoughts and your experiences.

Friday, December 21, 2012

A few odds and ends

Today I have included a few odd bits of interest. The first is a crossing I did using Gomesa flexuosa (syn. Oncidium flexuosum) and Oncidium sphacelatum. I crossed the two species because I wanted to see if the hybrid would be more cold tolerant but with more flowers. The seedlings are growing like mad! The hybrid is registered with the RHS as Oncidesa goldiana. I hope to flower it in the next few years.
Oncidium sphacelatum
Gomesa flexuosa
Oncidesa goldiana
The next is a photo I just took of the Euliophia speciosa seedlings I kept from the first batch of seeds I did in 2010. They are doing well and getting quite large now. Their pseudobulbs are underground, fat and healthy. The foliage is rigid and thick. The seedlings definitely benefit from a shallower tray with sandy substrate. They also enjoy heavy waterings between short periods of drying off.
Eulophia speciosa
The last photo was taken by Richard King of one of my flasks of Dendrobium harveyanum which contains a seedling that had flowered in vitro! This is not unheard of but it is not a common occurance.
Dendrobium harveyanum (photo by Richard King)

Thursday, December 13, 2012

Returning to tissue culture

I mentioned previously that I had decided to return to tissue culture at home, although space and TIME remains a serious luxury. As many of you will be aware, I joined the Walker Bay Orchid Society here in Hermanus shortly after I relocated here. The area is a biodiversity hotspot and boasts many different species of native terrestrial orchids. Some of these are endangered, especially those in the low-lying regions that are subjected to urban sprawl, farming and the spread of alien vegetation.
I have been in discussion with one of the members of the WBOS about our involvement in conservation of these local species which was briefly mentioned at the last AGM. Of particlar concern is a small local colony of Disa hallackii in Vermont. This species is considered one of the most threatened of all South African orchids and is currently listed as an endangered species on the SA red data list. We have approached the Vermont Conservation Trust through Mr. Duncan Heard who forwarded our correspondence to various role-players in Cape Nature and local government. We proposed to culture seeds in vitro collected from a known source and to raise seedlings that could be acclimated and returned to other localities under the control of Cape Nature. I have offered my time and my skills to do this free of charge in support of this species' local conservation. I believe in taking action, not just sitting around a few remaining plants in the wild and hoping they will remain there for my young daughters to see one day. The reality is that this species is in decline and if we don't mitigate the threats to this species by utilising our collective knowledge and skills I fear that it will become yet another memory of failed hope.
We are still waiting for a response to our proposal from all those recipients. In the mean time Patrick Donnelly and I have been actively looking into the permiting requirements which will allow us to proceed. I have ordered and received the media for the work and I have begun to optimise an experimental recipe using other terrestrial orchids from my own collection. One of my recipes was also recently used by Richard King for Disa uniflora which he found to provide significant shoot growth.
Lets hope that this story carries a happy ending...

15 December 2012: Follow up

Today I went to the plot in Vermont with Patrick and Dot Donnelly to see the Disa hallackii for myself. I felt an overwhelming sense of priveledge being there and seeing the few remaining plants in the entire area. The last count was 17 individual plants. We only counted 10 in the whole time we were there. What's worse is that this plot of land IS going to be developed and stands are already demarkated for several houses to be built. Much rubbel has also been dumped on this site close to the road. The majority of the area was previosuly covered by Port Jackson, which have since been cut down. However it is clearly evident that the remaining Disas are only found in the clear areas where the Port Jackson had not yet invaded and this is only a thin band! Since 19 November when my intial request for support went out to various role-players, I am disappointed to report that I have had no response from anyone yet - nearly a month later. C'mon CapeNature and local government! Please get involved! If you can't see the value in acting and acting NOW, then how do we maintain our faith in you and what you are mandated to protect?