| |
Introduction To Pleione Orchid Flasking
The equipment and procedures described here are for asymbiotic (no fungi) in vitro propagation usually knowing to amateur enthusiasts as orchid flasking. Symbiotic flasking using special fungi is another option sometimes used for other types of orchids but is an unnecessary complication for Pleiones.
The procedures described are all able to be performed in a normal family home, usually the kitchen, and the equipment and materials are all available or obtainable without spending a fortune. No professional knowledge or experience is necessary but meticulous planning, organization, timing and attention to detail is needed. The process is long with numerous stages where things can go wrong. Some stages are fiddly and need some dexterity. By far the most important factor throughout is good aseptic technique and preventing the interior of the flasks becoming contaminated with fungi or bacteria.
We aim to achieve the following: 1. Prepare gel media optimized for Pleione germination and growth.2. Prepare suitable containers (flasks) containing the correct amountof media.3. Sterilise the media in the containers using an autoclave(pressure cooker).4. Make a suitable glove box or laminar air flow cabinet for maintaininga sterile environment during sowing.5. Disinfect (surface sterilize) the seed immediately prior to sowing.6. Germination. Incubate the flasksunder optimized conditions oflight and temperature.7. Reflask into new media at certain stages of growth.8. Deflask and transfer immature plantlets into bark or moss media.9. Grow on to flowering plants.
Please note that my flasking technique is for producing bulk amounts of Pleione species to help protect Pleione in the wild. For flasking new hybrids you may wish to do an additional reflask and keep for another year before deflasking. When flasking the quantities I am currently doing it’s not practical to reflask for a second time due to the volumes of bulblets involved.
You can use commercially prepared media or make your own. Unless you have a good working knowledge of chemistry, biochemistry and plant nutrition and have access to chemicals and biochemicals then I strongly recommend purchasing and using prepared media. Companies such as Sigma/Aldrich and Phytotech supply a range of well researched media and other products developed specifically for in vitro orchid culture. Orchid maintenance media (Sigma P6668, Phytotech P668) at half strength is ideal for sowing Pleione seed. I buy the versions containing all nutrients including sucrose plus charcoal and other chemicals but excluding the agar which I buy and add separately at the recommended rate (approximately 7 grams of agar per litre of half strength media). A major advantage of this product is the inclusion of a pH buffer so that you don’t need to adjust the pH (acidity/alkalinity) with acids or alkalis and don’t need to use a pH meter.
Accurately weigh out the recommended amount of media (half the weight recommended for making full strength orchid maintenance media). I use a small portable jeweler’s electronic balance which is ideal and inexpensive. Place 1 litre of cold distilled or deionised water (Halfords sell it) in a jug. Sprinkle the media in while stirring vigorously. Keep stirring until the charcoal in the media has fully wetted and dispersed. Then weigh out 7g of agar and stir that in also. The media is then ready for adding to your flasks. Only make up enough for immediate use. Discard any that is unused as it will not store in this non sterile pre-flask state.
Traditional glass honey jars have now been superseded by clear polypropylene microwaveable food jars. Unlike many other plastics, polyprpylene is fully autoclavable (pressure cooker proof) so is ideal for the task. These clear flasks are available in various sizes, are cheap to buy and very light and easy to store. The smaller sizes are best suited for germination purposes. For later reflasks a small hole about 1 mm across need to be pierced in each lid and covered with a stick-on PTFE flasking patch to facilitate sterile ventilation but this is usually considered unnecessary for seed germination flasks.
While continually stirring or swirling the prepared media in the jug, to prevent the charcoal settling, pour small quantities into each flask. I recommend a depth of at least 1 cm. Lightly rest the lids on each flask with a small twist of aluminium cooking foil placed to prevent the lids sealing during autoclaving or when cooling (see picture). The flasks are now ready for sterilization in the pressure cooker or microwave oven. A pressure cooker will reach much higher temperatures than a microwave oven so is more efficient at sterilization. Some people have reported good results just using a microwave oven.
Stack the prepared flasks in the pressure cooker or microwave making sure the twists of foil are preventing the lids sealing. In a pressure cooker plastic flasks will float and tip as you load them so you need something to raise them out of the water. I use a coil of small diameter copper tubing placed as a platform in the bottom of the pressure cooker. Use lightly compressed balls of foil to pack the columns of flask to prevent tipping. Attach the lid and bring to full temperature and pressure. Maintain for 20 minutes and allow to cool and depressurize. Remove the flasks when they have cooled enough to handle with rubber gloves. As you remove the flasks pull out the foil twists and seal the lids. Be careful not to seal the lids too soon while the flasks are still very hot and full of steam or they will collapse when cooling. Place immediately on a cool window sill and do not disturb them again until the agar has fully set. Your flasks can now be stored until use. I recommend you keep them dry in a clean plastic bag in a dark cupboard safe from dust as the outer surface will need to be quickly sanitized again when flasking the seed.
Glove box or laminar flow cabinet
For flasking purposes you need nothing more complicated than a simple homemade glove box. Laminar flow cabinets are useful for more serious tissue culture work and are also great for flasking if you have access to one, e.g. if you work in a lab or you attend a college with such facilities. I personally don’t think the cost of purchasing or making a laminar flow cabinet is justified for home orchid flasking. I very successfully use a homemade glove box made very quickly and simply from a plastic storage box on its side with a front made from clear vinyl plastic sheet which is attached with duct tape. It has two round holes in the vinyl for access covered with drop down flaps. I originally tried using arm length gloves sealed into the holes but this made access for equipment, etc, into the box very difficult. Experience has proved that completely sealing the box that way is quite unnecessary. After all we are not dealing with pressurized systems, deadly toxins/diseases or radioactive isotopes. Never the less rigorous asceptic techniques and procedures are essential.
Glovebox Cutout Opening Covered By Plastic
The inside surfaces of the glove box should be thoroughly disinfected before use. I spray with a 1% bleach (sodium hypochlorite) solution (suitably diluted Miltons or Domestos will do). I also mist the air in the flask with bleach and allow to settle before use. Everything you need for flasking should be dipped in or sprayed with bleach before being placed ready for use in the box. It is worth cautioning at this point that you need to be wearing old clothes and shoes and a full length apron for flasking as you will inevitably end up with white spots everywhere. You also need to wear good quality surgical gloves and dip your hands in bleach before re-entry every time your hands leave the box during flasking operations. Bleach will be sloshing around and dripping everywhere so be careful. Use hair and eye protection. Keep the flaps closed when not in use.
If you have access to a microscope it is worth checking the seed for embryo's or you could waste time and money sowing seed that is not viable. I usually sow seed as soon as the pod is taken from the plant but have used seed that is twelve months old. Older seed does seem to have reduced germination and I therefore recommend sowing as soon as possible.
Few or no embryo's / 50% embryo's / 95%+ Embryo's
The seed must be disinfected (surface sterilized) to eliminate bacteria and fungi which would otherwise grow very rapidly on the agar medium. We do this by treating the seed with 0.5% sodium hypochlorite for at least 20 minutes. This also helps to break down any germination inhibitors that may have developed during drying and storage although this is less of an issue with Pleiones than with most other terrestrial orchids.
The procedure that I have found works well for me is to place a small amount of seed in a 15 ml test tube. Add 10 ml of 0.5% hypochlorite solution. Seal with a stopper and shake well. Incubate for 20 minutes with frequent shaking then allow to settle for a few minutes. Dip the whole tube in hypochlorite to sterilize the outer surface and stand upright in the glove box for a few minutes. The disinfected seed floats to the top where it can be scraped up the side using a spatula with a curved tip which roughly matches the curvature of the test tube. Don’t bother to rinse the seed. Just transfer it direct to the agar and spread it over the surface. The agar will rapidly react with and neutralize any excess bleach. Make sure the lid of the flask is only lifted as briefly as possible and that the inner surface of the lid always faces downward and does not touch anything. Seal the flask and remove from the glove box. Remember to label it. I normally disinfect and sow dozens at a time which needs careful preparation and timing. An assistant to sterilize and pass equipment and do the labeling, etc, as in an operating theatre is a great help.
Incubate the flasks somewhere with good light and room temperatures. A north facing windowsill in a centrally heated house is OK. I use special blue/red LED lights in a temperature controlled cabinet. Fluorescent and tungsten lights also work but may make it more difficult to control the temperature. I germinate with 14 hours light, 10 hours dark using a switch timer.
By using a low power microscope or high power magnifying glass I usually observe the embryos swelling and then changing colour within two weeks. The embryo first goes from white to yellow and then gradually green. Sometimes it happens within a few days. Occasionally it takes a couple of months. If no germination is apparent after 3 months then it’s probably not going to happen.
Eventually the embryos grow into little pointed protocorms complete with rhizoids. The best time to make the first re-flask is when the tips of the protocorms start elongating into a leaf although it can be done later.
The medium I use for reflasking is 75% strength orchid maintenance medium to each litre of which I add 50 ml of fresh coconut water. I sometimes also add 25 ml of fresh pineapple juice but this has the drawback of needing to adjust the pH with acids and alkalis and pH meter because the acidity of the juice overpowers the capacity of the pH buffer in the medium. If the mood takes me I may further add some benzyl adenine, an artificial cytokinin that boosts growth although coconut water and pineapple juice already contain natural phytohormones for that purpose. For reflasking I use much taller flasks and add the medium to a depth of at least 2cm.
The medium and flask sterilization and glove box procedures are as previously described.
If the transfer is done when protocorms are still very small then I just scrape them directly off the surface of the old agar and tip them onto the new agar with a spatula and spread them as evenly as possible. This takes very little time which minimizes the possibility of contamination. If leaves have formed and grown quite tall I scoop out and transfer small bunches. I incubate under lights as before but change to 16 hours light, 8 hours dark to boost growth. The sides of the cupboard are lined with a reflective foil type sheet which is available from all good hydroponic shops!
Late June I transfer the reflasks to a cold frame outdoors to acclimatize to natural conditions. This frame gets good light but is protected from direct sunlight. As the days shorten and night temperatures drop in autumn the seedlings naturally go brown and dormant just like adult Pleiones.
At this point I deflask the tiny bulblets (1-2 mm across) and remove the agar, dead leaves and roots. The tiny bulblets are then soaked in fungicide and allowed to air dry without rinsing. When dry they are stored in a cold dark cupboard in my unheated garage until spring. To prevent the very small bulblets desiccating I keep them in small sealed plastic containers. These are usually the same labeled containers, after cleaning, that I germinated them in.
In February/March I plant the bulblets in seed trays using a compost made from fine bark, perlite and cocoa fibre. The trays are covered with clear plastic domes to maintain high humidity. These are incubated under lights at 12 hours light, 12 hours dark. The compost is surface misted daily.
They usually come back into growth quickly and soon look like trays of grass turf. Liberal use of fungicide spays may be needed to control damping off because of the high humidity and close packing of seedlings. In May I remove the covers and transfer to the greenhouse to be treated the same as other Pleiones propagated asexually from bulbils and small offsets. They can take 3-4 years to form flowering pseudobulbs.
|
.......................
|
© Copyright Koolplants, 2008. All Rights Reserved.