Sunday, August 29, 2010

Undifferentiated spikes can be influenced by temperature to produce keikis

Recently I increased the minimum temperature in the greenhouse from 18°C to 23°C to encourage new vegetative growth on my Phalaenopsis plants that I had used for cloning. Three plants remained with very early spikes that had been too young to cut for cloning. Two of these were still undifferentiated and the other was slightly longer with the beginnings of flower bud differentiation. The latter continued to grow in length normally with reproductive growth while the other two began to produce vegetative growth. It is well documented that vegetative growth in Phalaenopsis plants is triggered at around 26°C but it may be possible that the plant spikes when still very young and undifferentiated are influenced to produce vegetative growth when the temperature differential is reduced by increasing the minimum temperature. So, I can anticipate two additional plants… I intend to leave one of these on the mother plant to increase its size as a specimen plant. The other one I will remove when it is large enough and will pot it up.

Thursday, August 26, 2010

Update on Phalaenopsis pollination 26 August 2010

Pollinated Phal flower front view
It has been 12 days since the pollination of 3 flowers from the large pink Phalaenopsis from Woolworths (2 x P. Golden wonder X P. no id Woolworths; 1 x P. no. id Woolworths X self). Within 48 hours the flowers wilted. You can already see the transformation beginning. The back of the flower column is beginning to swell and elongate and the front of the column has enclosed the pollinia and stigma. The petals, sepals and labellum have all become dry and papery now. The literature suggests an average time to harvest of the green pod at about 5 months after pollination for large hybrids. This will be sometime in January and I will be keeping a beady eye on any pods. By this time I would have acquired some Sigma Phytamax P6668 culture medium for seed germination. I am still working out which gelling agent I should use though, and how much will be needed. I will probably use Sigma’s Phytagel but will see if I can find any other alternatives that won’t be too expensive.

Pollinated Phal flower side view
On the same subject but a different orchid, I stumbled across an abstract for a recent South African publication on “In vitro asymbiotic seed germination and seedling growth of Ansellia africana Lindl. by Vasudevan, R. and Van Staden, J. (2010) from the University of KwaZulu-Natal’s Research Centre for Plant Growth and Development. I subsequently requested a reprint which I kindly received from the authors. The study tested whether different concentrations of sodium hypochlorite (during sterilization of seed) influenced germination and success of seedling development. Ultimately their findings suggested that the different media used were probably responsible for their different results and that keeping the seeds in darkness for a period during germination improved the quality of the protocorms. They suggested that the darkness probably promoted rhizoid development of the protocorms which once exposed to light had a better supportive mechanism already in place for the transportation of nutrients from the medium while commencing with chlorophyll development. An interesting read indeed! They recommend using Phytotechnology’s P668 orchid maintenance medium (this is nearly identical to Sigma's P6668) for seed germination in this species. I wonder if they would let me have a seedling or two from the study… mmm…I will enquire with the authors. Here is the full reference to the paper:

Vasudevan, R., & Van Staden, J. (2010) In vitro asymbiotic seed germination and seedling growth of Ansellia africana Lindl. Scientia Horticulturae 123: 496 – 504.

Wednesday, August 18, 2010

Dendrobium kingianum first flower

I have attached a picture of the first flower of a Dendrobium kingianum specimen that was given to me just short of a year ago (thanks Kevin O!). It is an interesting colour. The most common form is a deeper pink and I have even seen a purple variant. There is also a pure white flower but this one has very pale pink flowers. The plant is still very young but is shooting new growths now as well from its base. Two keikis that were formed previously were removed and planted next to the parent plant. These are also doing well. Apparently, the trick to getting this species to flower and not produce keikis is to allow them a period where they can be kept dry. Too much water and the buds that form the flower spikes produce keikis.

Dendrobium kingianum

Sunday, August 15, 2010

Cross pollination 14 August

On Saturday 14 August I cross pollinated two of my Phalaenopsis hybrids, P. Golden Wonder with an anonymous large pink hybrid from Woolworths. I will systematically post the progress of the pods (if any) on a monthly basis. Here are the flowers. The pollen donor is P. Golden wonder.

P. Golden wonder
P. Large pink (Woolworths)

Wednesday, August 11, 2010

New plants arrive from Van Rooyen Orchids

This morning I finally received my plants from Van Rooyen Orchids. The plants include Phalaenopsis pulchra (species), Doritaenopsis Purple gem "Blue bird" (intergeneric hybrid) and Brassia rex "Otto" (hybrid?). All the plants are not in flower but they are all in good condition.

Brassia rex

Phalaenopsis pulchra

Monday, August 9, 2010

The first attempt: In vitro micro-propagation

My sterile transfer chamber
I had been looking forward to getting stuck into the tissue culture process the entire week! Friday finally came and I eagerly set about getting everything laid out that evening for Saturday and ensured that everything that I needed I had. That evening I mixed up a litre of Phytotechnology Labs’ orchid multiplication medium which is an agar-based and ready to use medium for encouraging vegetative growth of stem nodes. Once heated for just enough time to allow the agar to melt, I poured the mix into various jars and some McCartney bottles to be sterilised. All containers of medium and a bottle or two of tap water and my dissection kit were placed into my Fagor pressure cooker for sterilisation for 30 minutes. After the time was up I let everything cool down for a bit inside the cooker and then opened it up and moved everything into the sterile transfer chamber. The chamber had been pre-sprayed with a 10% bleach solution and had been irradiated with UV for 30 minutes. After everything had been placed inside I switched on the UV lamp again for a further 30 minutes considering that my hands were probably a source of potential contamination.

The next morning the media had cooled and set. I prepared two jars of bleach solution, one with 10% and the other with 5% to which I added 3 to 4 drops of Tween 20 from Phytotech. These jars of bleach solution were for the sterilisation of nodal sections of the flowering stems of my parent plants. As these jars would initially be used outside of the sterile transfer chamber, I immersed them in a larger container of 10% bleach solution to ensure that the outside of the jars would not pose a contamination risk. In addition, I prepared a third jar of Virkon S using a 2% solution. This jar too was immersed in 10% bleach to avoid a possible contamination source. Once this was done I set off to the greenhouse and selected my plants for culture. The flowering stems were cut and placed into a jar of water and brought into the kitchen for washing. Each flowering spike was thoroughly rinsed and washed under normal tap water and then cut into nodal sections. Additional sections of internode were also cut. All these cuttings were placed into the Virkon S solution and agitated while I prepared to sterilise my hands with alcohol-based hand sanitiser (the new one by Dettol). Once I was happy with my hands I put a pair of surgical gloves on and repeated the procedure with the hand sanitiser and lit a candle next to the chamber for flaming my tools.

Phalaenopsis nodal sections
Now I was ready to proceed and carefully lifted the trap door in front of the chamber making sure I did not touch anything unnecessarily. I unwrapped my dissection tools from their foil covering which I discarded and placed all the tools into a jar of ethanol inside the chamber. Next, all three jars (the bleach solutions and the Virkon S) were moved inside the chamber and the cuttings were carefully removed from the Virkon S using a large forceps into the 10% bleach solution for 15 minutes. During this time I frequently sterilised my gloved hands outside the chamber. After 15 minutes I removed all the cuttings and placed them on the surface of an inverted sterile petri dish that was inside the chamber. Before and between use, all tools were flamed frequently and returned to the jar of ethanol to avoid contamination. The nodal sections of flowering spikes were carefully lifted from the dish and using two forceps, the bract surrounding the dormant bud of each node was removed and discarded. The naked nodal sections were placed into the 5% bleach solution while the internodal sections were chopped into many small sections (about 1-2mm thick) using a sterile scalpel. Once these had been added to the 5% solution of bleach, the jar’s lid was closed and the cuttings sterilised further for 10 minutes with frequent agitation. After the 10 minutes, the nodal sections were again placed on the surface of the petri dish and the ends of the sections that had been damaged by exposure to the bleach solution cut off and discarded. Thereafter, these nodal and the internodal sections were placed into a jar of sterile tap water for rinsing. The jar was frequently agitated to remove traces of bleach from the plant tissue sections. Each nodal section was then carefully inserted (and correctly orientated) into the prepared media of individual McCartney bottles and the intermodal sections were placed into the larger jars of media as well as 2 multi well tissue culture plates. I am keen to see which containers yield the best results.

My incubator
All containers of plant tissue were tightly closed inside the chamber before being removed together at the end to avoid contamination. PVC electrical tape (insulation tape) was used to seal off the lids of each vessel and a label with a description of the contents and the date was added. The tissue culture multi-well plates were placed inside sterile zip-lock sandwich bags. Each vessel was then placed inside the incubator with a programmed light regime of 16 hr : 8 hr and a temperature of 26°C. The entire process was extremely time-consuming and it took the better part of the entire Saturday to complete for 4 different Phalaenopsis hybrids and a species, P. amabilis. The next day my curiosity got the better of me and I repeated the preparation of the chamber and completed some internodal and nodal sections of an Oncidium hybrid, Dendrobium kingianum and even experimented with the meristematic tissue of a new root growth on one of my Laeliocattleya hybrids! Now I just need to keep an eye out for any contaminants that might grow inside any of the vessels. I expect the odd one. Even during my PCR training a few years back you could never tell when you would get some contamination. Given the literature, it appears that an average contamination of 10% can be quite common.

Phalaenopsis internode
If all goes well then within a month or so I should start to notice the swelling of the nodes on the nodal sections and possibly some small leaves being formed. The internodal sections should produce callous-like tissue from which protocorm-like bodies should form. If I manage to avoid contamination then the next stage will be to move nodal sections into a rooting medium when leaves become evident. Internodal sections can be sub-cultured onto the same medium for the proliferation of protocorm-like bodies until plantlets appear. Here (I hope), larger quantities of clones can be produced through the process of sub-culture. Once any plantlets emerge, these will be moved to the rooting medium as for the nodal sections and cultured further in the incubator…

Here’s holding thumbs!

Tuesday, August 3, 2010

Recent arrivals, pollination and preparation!

Yellow Phal from Exotic Plant Company
A few weeks back I drove up to Franschhoek to visit Mike Tibbs' greenhouses at Moreson wine estate and bought a few Phalaenopsis species and hybrids. He had some really good looking P. amabilis available for a steal so I took two that were just finishing with flowering. I also took home a lovely large yellow hybrid and a large pink. The yellow hybrid will form part of my initial experimentation hopefully later on this weekend when I attempt my first stem propagations. I have been eyeing out a few of the dormant nodes on the flowering spike and might just get a few of these to grow... But, in the mean time I have also purchased two plants from Van Rooyen's Orchids,  a Doritaenopsis hybrid and P. pulchra, a species native to the Philippines. The former is a blue hybrid from P. equastris parentage so the flowers are small, but should hopefully be numerous.
Ansellia africana pod (1 month after pollination)
On 4 July this year I self-pollinated my large Ansellia africana, a monotypic epiphytic species (non Phalaenopsis) that is native to Africa and is found in South Africa in the North East and down the coast probably North of Durban. I was fortunate to get some plants from Zimbabwe a few years back which did very well down in the Cape. I have produced two seed pods which I will attempt to get some viable seed from. However, I have no idea how long the pods take to ripen and mature but I assume it to be several months. It is still early days but I have attached a picture of one of these pods which is now about 2 months old.

I have begun with my preparations to clone a few of my plants. To date I have constructed a sterile transfer chamber from Plexiglass (perspex) and installed a germicidal UV tube (20 watt) into the ceiling for sterilisation. Finding a suitable pressure cooker here in Cape Town was more difficult and I learned that not all pressure cookers being sold are rated to 15 psi. In fact, most offer a maximum rating of only 12.6 psi which is just not good enough for media sterilisation. After much searching I managed to find a Fagor pressure cooker, which is made of stainless steel and is rated to 16 psi. I bought it from Makro in Montague Gardens. Next I had an incubator built to my specifications by a colleague out of wood and glass and installed an extraction fan and a thermostatically controlled heater cable. The incubator has two glass shelves to allow adequate light from a fluorescent tube inside to penetrate to 2 levels of flasks. Buying orchid media for cloning and/or seed culture can be expensive. To my best knowledge no media are commercially available here in South Africa for orchid culture although most local breeders probably have their own international contacts or personal recipes. I had to purchase my culture kit including various media directly from Phytotechnology Laboratories in the USA. As a result, the courier charge was more than the cost of the contents but everything arrived safely to my door within a week after placing the order. There is an African distributor based in Kenya who I originally dealt with but I had a terrible experience with them and their lack of professionalism and poor customer service so I opted to place my order directly with the producer (Phytotech) who were extremely efficient.

So, now I await the time (hopefully on the weekend) to get stuck in and test everything out.

Monday, August 2, 2010

To begin with...

I have set up this blog to document my exploration of species and hybrids of the genus Phalaenopsis, the moth orchid.

Peloric yellow Phal in recovery
My love affair (rather addiction) with orchids began when I was in my early 20s. My first orchid was a large white Phalaenopsis hybrid that lasted about two months before I killed it. Looking back at this now I realise that I just "loved" it too much and probably over watered it. However, while it lasted it was wonderful. Since then I have kept and maintained many orchid varieties but Phalaenopsis have remained my absolute favourite. In 2003 my then rapidly growing Phalaenopsis collection drove up with me from Cape Town to Durban where I stayed for a further 2 years. In this time my plants grew and bloomed like never before and when I finally returned to Cape Town in late 2005 I did not have the heart to bring all of them back with me. These ended up with willing foster parents and to my knowledge they are still doing very well. I did keep just a single plant, my only peloric yellow hybrid that I had nurtured from a yearling I bought some years back in Stellenbosch from Else Hall of then Stellenkloof. This plant ended up with my aunt in Port Elizabeth (PE) on my way down to Cape Town (Why? - I still don't know). In June this year at a family gathering in PE I came accross what was left of the once large and healthy peloric. It had been moved outside and the main plant had died. A single lateral bud had developed 2 small stunted leaves but there were no roots. I bundled it up into a plastic container lined with damp tissue paper for the drive back home where I trimmed off all the dead bits and re-potted the tiny remaining bits of life into milled bark. I placed it on my desk with an architect's lamp positioned over it for adequate light and warmth. An old aquarium thermometer positioned next to the plant provided an indication of temperature. Two weeks later and the second leaf had begun to expand. Two months later and a third leaf had emerged and a new set of roots. I was happy.

Recently I set up a greenhouse (kit-type polycarbonate twin-wall) dedicated to my orchids. After some fiddling I got the insulation and heating right. Growing happily inside is my precious peloric which I hope to get back to flowering size within a year or two.