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!


2 comments:

  1. Shoe Dave,what a process! I know that all your hard work and effort will pay off. Can't wait for the next chapter!
    Tersia

    ReplyDelete
  2. Virkon


    Great job done by author. Information helps me a lot to improve my skills. Thanks for the author for sharing. Please keep posting.

    ReplyDelete