More greenery

With this year drawing to a rapid close it is only fitting that I add some more images of some more germinating seeds and protocorms. Things in the incubator are getting rather crowded now and I will have to build a new unit in the New Year to accommodate all the re-plates! Some of the existing protocorms are developing well and I now have protocorms of Disa uniflora, D. atricapilla, D. thodei, D. harveiana, Ansellia africana, Eulophia speciosa, E. angolensis, Acrolophia capensis, Polystachya ottoniana, Dendrobium kingianum X speciosum, Dendrobium delicatum X speciosum and Stenoglottis fimbriata. The Dendrobium hybrids are from a friend's seeds that I sowed for him from green pods recently. These will need to be transflasked soon since they are a bit overcrowded.

In addition, the Phalaenopsis hybrid seeds are swelling nicely and there is no contamination. I have also since sown some more terrestrials but on a 1/2 strength MS medium supplemented with 10% coconut water (extremely hard to find in South Africa!), vits, carbon, glycine and insitol. I am interested to see how protocorm development differs from that of the same species sown on BM-1. Although I got good germination with BM-1 I am a little concerned about the lack of Nitrogen source as compared with that of MS. I hope to compare notes here and will keep you posted.

Ok, here are some images.

Ansellia africana

Dendrobium hybrid

Dendrobium hybrid

Dendrobium hybrid

Polystachya ottoniana

Phalaenopsis hybrid green pods sown

After waiting for 110 days, the first two Phalaenopsis hybrid green pods were ready to be sown using the green pod method. I ceremoniously cut them from the mother plant last night and sterlilized them in sodium hypochlorite (Jik straight) for 10 minutes before rinsing and flaming in ethanol. The pods were cut in half and then split laterally with a sterile scalple and four flasks of seeds were sown from each pod (see below). Now we wait to see if there will be any success!



Phalaenopsis hybrid progress to 110 days after pollination




Seed pods and seeds sown



Viable seeds with embryos

Disa draconis - conservation begins at home

I spend my time driving to work in the morning thinking about various issues that pop into my head. Recently, one of these issues has been the plight of some of our South African terrestrial orchid species (geophytes). I has recently read a conservation update on the state of the remaining population of Disa barbata and I was pleased to read that some seedlings that had been reared in vitro with the help of Hildegard Crous had been made available to some specialist Disa growers. I have always believed that private collections have an inicredibly important role to play in the support of species preservation. Disa barbata is maybe just the tip of the ice berg and I wonder how many other currently endangered and threatened local species will also end up one day known only from a single population in a single locality? If we consider the impact of urban sprawl on natural habitat and the invasion of alien trees and other flora and fauna, how long will it be before we are faced with a similar situation with many other species? One of these species could possibly be Disa draconis which was once thought to be common until separated into three species; D. draconis, D. harveiana and D. karooica in 1995 by Johnson and Linder (Johnson, S.D., & Linder, H.P. (1995). Systematics and evolution of the Disa draconis complex (Orchidaceae). Botanical Journal of the Linnean Society, 118: 289-307). The authors mentioned that D. draconis is now restricted to only a few highly threatened populations on the West coast. Since this article was published 15 years have passed...

In an e-mail to Hildegard I mentioned to her that I had germinated D. thodei, D. uniflora and D. atricapilla and I was curious as to the in vitro requirements of D. draconis for which I had some seeds. She replied that the medium used for D. atricapilla would probably be the one to try for D. draconis seed germination so last night I sowed some D. draconis seeds on this as well as dual-phase Disa media. I was also curious to see if the seeds were viable and if they had embryos. I estimate the seeds to have about 70-80% embryos. Attached bellow is a photomicrograph of one of these seeds.

So, now we wait and see if this little experiment will work. It will be a good investment of time and I hope to report positive findings along the way over the next few years.

Disa draconis seed with embryo

Clones and germinating seeds!

What a pleasant beginning to the week. The weekend was particularly eventful and much of my hard work and patience with my second run of clones and various orchid species seeds has paid off.

The first batch of Phalaenopsis clones did not do well at all and I worked out through a process of elimination that the problem was the lack of ventilation. This appears to be crytical in their development. The second run is working better. I have since made my own medium, having bought the various constintuents and this seems to be working just as well as the formulation sold by Phytotech Labs. I will however keep adjusting my formula to optimise growth as I go. In the mean time I have attached a photo of a P. amabilis clone producing leaves.

Phalaenopsis amabilis clone

While working hard on getting the clones to work I had also experimented with various orchid species seeds which I had flasked using various media including Sigma's P6668, BM-1, Murshige and Skoog and a modified version of the dual-phase Disa medium of Thompson et al. (2006). After waiting for what has seemed like forever, I finally have some images to post of my relative success. I say relative because it is still early days and I have a lot more seeds in vitro which await germination. Anyway, it is most exciting to get some positive results finally and on 12 December I will also get to sow the Phalaenopsis seeds that have been patiently maturing on my plants for the last 108 days.

Disa thodei germinating

Disa uniflora germinating: note shooting protocorms

Polystachya ottoniana germinating



Ansellia africana germinating



Stenoglottis fimbriata germinating

Incubator full of seed flasks!

Rescue operation and another interesting mould

Recently I had been keeping an eye on a creeping baterial contaminant on one of my plates of P6668 Phalaenopsis gigantea seed cultures. I decided to rescue a section of the unaffected seeds by cutting them out and placing them into a new sterile dish. It worked and the seeds are still doing well without any further contamination (see pics).



Bacterial contaminant and unconatminated part removed

 


Uncontaminated part with P. gigantea seeds





Additionally, I have included an image of a contaminated plate of P6668 with another interesting mould. This one had no seeds and was the only one of 16 plates of a new batch of media that became contaminated. Good for the records.



Another interesting mould!



Sowing Ansellia africana seeds

Ansellia africana pod harvested at 116 days old

I had been keeping an eye on my Ansellia africana seed pods that had been slowly maturing on the mother plant for just over 3 months. I noticed that the first pod began to turn yellow quite rapidly at 96 days old so I harvested it a few days ago and sowed the seeds on P6668 half and full strength supplemented with sucrose. The other pod seemed to remain green so I thought I would leave it for as long as possible but it turned yellow yesterday and I had to harvest it as well. This pod's seeds were sown on the same medium yesterday evening. The problem however is that literature that I had consulted suggests that A. africana pods take up to a year to mature and that green pod harvesting can be done at 5-6 months. My second pod was only 116 days old so I wonder whether the seeds will be viable. I can only guess that the constantly high temperatures in my greenhouse sped up maturation, well I am hoping this is the case. The mother plant is certainly not stressed and is growing like a weed and has shot out 7 new shoots so it is happy. I have included below images of the yellowing pod just harvested and one of the flasks of seeds.

A. africana seeds innoculated on P6668

Bacterial infection wipes out my flasks!

Usually within 3 days after sowing or any flasking work, contamination can become visible. I unfortunately have lost most of my new flasks of Phalaenopsis seeds to a bacterial infection. Looking for a possible cause I can only think that the filter paper-method coupled with short sterilisation time was the cause. All is not lost however. I have learned a valuable lesson here - bugger the paper method! I am going to upgrade to automatic pipette and autoclavable eppendorf tubes and tips! I will get these on Monday. In addition I have requested more seeds of the same species that were given to me previously and these will hopefully give better results after the amendment of my sterilisation method.

I have included some images of the flask contamination.

Fungal contamination - not the Phalaenopsis seeds though

Bacterial contamination of the Phalaenopsis seeds

in vitro part 2: seeds seeds seeds!

I was most fortunate to receive seeds of the following Phalaenopsis species (click on the names for links to images): P. cornu-cervi, P. aphrodite, P. gigantea, P. taenialis, P. lindenii, P. celebensis, P. violacea forma mentawai and P. heiroglyphica. Many of these species are certainly difficult if not impossible to get in South Africa so I am most pleased to have the opportunity to hopefully raise some by seed.

I purchased some Phytamax P6668 and P1056 from Sigma in anticipation of the seeds and for later re-plating respectively. A colleague of mine uses P6668 at half strength with added 20g sucrose so I prepared two sets of flasks with both full strength and half strength P6668 with added sucrose (10g and 20g respectively). I finished my media preparation and sterilisation on the evening of 11 October and  I did not have the time to work though all of the above species but did flask P. gigantea, P. cornu-cervi, and P. violacea forma mentawai. I also threw in some Microcoelia exilis for good measure but have no idea if the tiny pods were carrying any viable seed... we will see. The rest of the species were done on 12 October along with some Disa crassicornis and D. atricapilla.

I must admit sitting down in front of the sterile transfer chamber on the first night with all these species was quite a rush! I had one chance to get it right and hopefully I did not get any contamination. I followed the advice of my colleague who said I should sterilise the seeds with 0.5% sodium hypochlorite (active %, not v/v concentration) for only 2 minutes before rinsing and sowing. This to me seemed a bit short and in the back of my mind I am hoping it was long enough to inactivate any potential bacteria and fungal spores. He has done this before with good results though so lets have faith. I should know within 3 days or so and will either be sadly disappointed or happy with the results. I certainly hope all goes well.

I thought I would also add a brief update on my Phalaenopsis hybrid seed pods that are progressing very nicely. The selfing of the large pink from Woolworth and the coss with P. golden wonder are half way now. They will be harvested in mid December. Other pods are at varying stages of developemnt and I have had good success with most crosses with only a few rejections.



Phalaenopsis seed pod - 2 months on



Serendipity: Stenoglottis longifolium

I was recently on a mission to find some local terrestrial species and/or seeds of these for a colleague in Germany with whom I am doing a seed swop. I had been promised some Phalaenopsis taenialis, P. cornu-cervi, P. lindenii, P. celebensis, P. aphrodite, P. violacea (forma Mentawai), P. gigantea and P. heiroglyphica. Finding local species' seeds is not easy! I drove down to Somerset West and visited the Helderberg Nature Reserve's indigenous plant nursery and found two small Stenoglottis longifolium plants, one of which still had an old flowering stem attached with two seed pods. I removed the stem and the two pods for my colleague and I have included an image of one of the plant below.



Stenoglottis longifolium



Visit to Duckitts orchid show 16 September 2010

Yesterday (Thursday) 16 September I took my family up to Darling to the opening day of Duckitts' orchid show on Oude Post farm. I hadn't been at the show for several years now so I promised myself that I would be there early on the first day to ensure I had a good chance of getting the plants I wanted from the sales area. To be honest I was a little disappointed with the range of plants on sale but also the fact that the Phalaenopsis on sale did not bare any labels, so no grex names or parentage. I queried this with the volunteers servicing the sales area but they could only suggest that these plants had been produced for the pot-plant trade and therefore were devoid of any discernable nomenclature. Well... I ended up buying some great looking plants anyway because the flowers looked particularly high-quality. All these were re-potted into bark chips as soon as I got back home. I also bought a flowering Angraecum sesquipedale (var. angustifolia) and I had been hoping they would be on sale. There are lots more but none in flower.

I have compiled a few photos that I took randomly while wondering through the show area. There were some really beautiful plants on view some of which I could not find labels for to provide you with any names, but I will do my best...I have also added images of those which I bought - enjoy.

Mini Cymbidium something or other

A deep red-brown Cymbidium

My Angraecum sesquipedale (var. angustifolia)

Ansellia africana pure yellow

Ansellia africana heavy spots

Epidendrum pseudoepidendrum

Dendrobium sp.

Disa hybrid

Epidendrum something or other

Aerangis hyaloides

Oncidium someting or other

Laelia? something hybrid

Large Cattleya intergeneric hybrid something

Phalaenopsis one I bought

Phalaenopsis one I bought

Phalaenopsis one I bought

Phalaenopsis one I bought

Update 10 September 2010

Ansellia africana

My A. africana parent plant flowers

Prof. van Staden from the Research Centre for Plant Growth and Development at the University of KwaZulu Natal has kindly donated a few ex-vitro A. africana plantlets from his research. A friend of mine in Durban is organising to collect the plants and ship them down to me next week. These are completely yellow flowers, not the mottled kind and are from KwaZulu Natal. I am looking forward to receiving them.  My pod-parent plant recently flowered again so I took some more photos and photos of the progress of one of the pods. Both pods are doing very well.

As above (close up)

A. africana pod as of 10 Sept 10

Phalaenopsis Woolworths big pink pods

One of the three pods did not survive (P. Golden wonder X P. Woolworths big pink) but the other two (P. Golden wonder X P. Woolworths big pink and P. Woolworths big pink X self) are growing nicely and developing well.

P. Woolworths big pink pod as of 10 Sept 10

Stem props / clones

After initial contamination of some of the containers, most stems are doing well but growing very slowly! Its worse than watching paint dry! Some stems did not develop at all and have died which is to be expected from plants that may contain contaminants within the stems themselves. I have moved the P. amabilis stems into separate McCartneys into new media. The Oncidium stems have (all but one) produced flower spikes from their nodes... this is odd but not unique. I will sub-culture these further when they have matured. All (?) the internodal sections appear not to have done much at all. I will keep an eye on them anyway for a few more months but by now something should have happened so I think they may not make it. This is probably due to the concentrations of Benzylaminopurine and/or Naphthaleneacetic acid. I will re-look into this again later.

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.

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.