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