GROWING TILLANDSIA SEED USING ARTIFICIAL MEDIA AND STERILE TECHNIQUES

By: Dr. Gordon Slack

Anyone bitten by the plant bug logically wants to have more plants, and growers of Bromeliads are fortunate in that nature takes care of most of our wishes by the natural pupping process of our plants. However, those of us who have a really bad infection from this bug are usually not satisfied with the one or two pups our plants produce to maintain our plant collection and to swap with our friends. We succumb to the desire to have as many good plants as possible, so we start dabbling in growing Bromeliads from seed. With Pitcairnioid and Bromelioid seed there is usually little problem and satisfactory germination and growth occurs on a number of different moist growing media. With Tillandsioid seed, nature has designed a different dispersal mechanism such that only a few seeds land on a suitable substrate in a favourable growing environment. The Bromeliad literature is full of methods designed to improve on nature and provide ideal conditions for Tillandsioid seed growth and my first trial was the twig method of Richard Oeser. Unfortunately I was not able to achieve "complete success in raising tillandsias from seed" using this method and other methods tried over a period of a couple of years were equally unsuccessful.

Obviously a number of people have succeeded in growing tillandsias from seed as demonstrated by the recent article of Mark Dimmitt in the Journal of the Bromeliad Society and the presentations at Bromeliads III in Brisbane in 1985. About six years ago I was bitten by the orchid bug and after successfully germinating and reflasking my first orchids using aseptic cultural techniques, I felt that similar methods would be ideal to provide a long term growing environment for tillandsia seed. This has proven to be the case and I would like to demonstrate this method to you. Some background reading was necessary and some of the chemical and botanical terms were unfamiliar. Obviously modifications are necessary to adapt methods used in the massive orchid factories of Taiwan to our kitchen table and window sill.

Unlike orchids, bromeliads, on germination, are capable of producing a plantlet with chlorophyll to synthesise sugars for the plant's energy needs. By providing a sterile environment with adequate humidity, the effects of casual pathogenic fungi or predatory insects are overcome. The minute amounts of chemicals needed for the growth of cell walls and the production of protein and sugars in these cells is provided by the growing medium which is based on agar - a seaweed extract with physical characteristics similar to gelatine.

A number of formulae for the composition of media suitable for the germination of orchid seeds have been published - the standard probably being Knudson's "C". Benzing suggests the use of a soluble fertiliser in dilute form mixed with agar and I have tried on one occasion a mixture of hydroponic fertiliser and a small amount of fish emulsion in agar and this gave satisfactory tillandsia seed germination, although growth rate was slower than with the G and B orchid seed growing or replate media I normally use (obtainable from BACTO LABORATORIES). These scientifically formulated mixtures are provided in containers of either 35gm or 500 gm and the bulk powder should be weighed out in quantities of 35gm, the amount necessary to make one litre of growing medium. This is sufficient for about twenty small bottles which provide enough surface area for the germination of about one hundred tillandsia seed in each.

PREPARATION OF THE GROWING MEDIUM:
Clear glass or sterilisable plastic containers are used and any small glass bottle with a wide neck, such as those holding fruit juice or cordial, is satisfactory. Most of my bottles come from hospitals and have contained X-ray contrast or anaesthetic solutions and anyone with access to these will find them of suitable size and the rubber bung is easily pierced. Whatever bottles are used, they should be thoroughly cleaned and a tight fitting lid or rubber bung inserted. As germinating seeds and plantlets need oxygen and carbon dioxide, air must be able to enter the bottle without bringing with it bacterial or fungal spores. A breathing hole can be punched out of the lid and filled with cotton wool or in some types of glass bottles or test-tube agar slopes, a wad of cotton wool is placed in the neck of the container and covered with several layers of aluminium foil. The growing medium is made up by adding the appropriate weight of agar and nutrient chemicals to one litre of distilled (I use de-ionised ironing) water while it is gently heated and stirred constantly until dissolved.

Lids with breathing holes are placed on each container after an appropriate amount of solution is added to provide a half to one centimetre thick layer of jelly. This growing medium must now be sterilised to eliminate fungal spores and bacteria which have probably fallen into the solution during its preparation. The most efficient sterilisation is achieved by using a kitchen pressure cooker with a pressure of fifteen pounds per square inch for ten to fifteen minutes. Other methods of sterilisation are described, usually consisting of repeated boiling of the solution and yet another method described for sterilisation of orchid growing media incorporates hydrogen peroxide in the growing medium. I have no personal experience of this.

After the pressure cooker cools and its lid is removed, the bottles containing the growing medium are removed and placed on their side to allow cooling and solidification of the jelly. It is important that the layer of jelly does not reach the neck or the breathing tube in the lid. Only a thin layer is necessary to provide adequate plant nutrition. Re-sterilise any containers which have blown their top during the pressure cooking process as spores may re-enter once the pressure cooker is opened. Bottles can be kept for a period of several weeks or months or used as soon as the agar cools and solidifies. I prefer to leave them for a couple of days and any contamination usually becomes visible within this period.

PREPARATION OF SEED.
Most tillandsia seed would be contaminated by the usual airborne fungal spores by the time we harvest and prepare to sow it. Using the normal methods of seed germination, we depend on local fungicides and the creation of conditions unfavourable for fungal growth to prevent these spores germinating and fungi causing plant death. If growth is to occur in a sterile environment this surface contamination must be eliminated. The method used has been gleaned from a number of different orchid references, but involves first soaking the seed in a small quantity of sugar solution, one teaspoon per cup, for twenty four hours or so (a few drops of detergent or surfactant solution help the wetting of seed and hairs). This encourages the germination of fungal spores and makes them more susceptible to the later chemical disinfection, which is usually achieved with household bleach in about fifty/fifty dilution with water, although I have used pure bleach and I have also achieved seed disinfection with hydrogen peroxide. Theoretically this should be best as it oxidises to water and in the growing flask would not leave behind any chemical residue which could harm growth. However, sodium hypochlorite bleach has not caused any problems to my knowledge in actual seed germination.

SEED SOWING:
The area where the seed is transferred to the sterile growing medium obviously should also be sterile. This, in a large commercial concern, is achieved by using laminar flow cabinets with air sterilisation by ultra violet light. On the kitchen table it can be done in a plastic bag, again using bleach for surface disinfection. Some tools are needed to physically transfer the seed and my most commonly used one is a pair of angled tweezers, a stainless steel wire loop or hook embedded in a glass handle and sometimes an old broken dentist's pick. These can be sterilised in a flame and cooled in a bleach-soaked cloth before transferring into the plastic bag or simply sprayed with bleach and wrapped in a clean, bleach-soaked cloth. A clear plastic bag big enough to contain the number of bottles required is used for the transfer chamber and after the tools, the seed container and the growing bottles are transferred into the bag, the inside of the bag is liberally sprayed with full strength bleach solution and a rubber band is placed around the open end of the bag.

After washing my hands thoroughly I spray my right hand and part of the forearm with bleach solution, although it would be more appropriate to wear a glove and spray the glove with bleach solution and insert it through the rubber band closure so that inside the bag we now have a container of seed, a bottle containing our growing medium, some cloths, a number of tools to transfer the seed from one bottle to the other, and a right hand. Through the clear plastic these procedures are carried out under direct vision and the left hand outside the bag is used to hold bottles, instruments and generally manipulate things so that the transfer can be achieved as rapidly and efficiently as possible. Even after spraying bleach into the bag, there is a possibility of contamination still existing. The risk of transferring this to the growing medium is greatly reduced if the growing bottles are laid horizontally on the work surface, a small vertical opening is presented for seed transfer and the growing container is open for as short a period as possible.

The stopper is placed on a bleach soaked cloth - the forceps or a hook is used to pick out the mass of seed with its tangled hair appendages and transferred to the growing medium. A very short time is spent trying to spread out the seed as much as possible before the growing bottle is re-sealed. In Benzing's description of this process, he suggests removal of the coma hairs from the seed and having attempted to do this on a few occasions it is a most tedious process and does not seem to improve germination. It probably does not greatly decrease the chance of contamination. Initially I spent a great deal of time trying to separate the mass of seed in the belief that a tight wad would not germinate freely. This has proved not to be the case. I usually work with batches of about six different bottles at a time so it is most important before putting the growing bottles and the seed bottles in the plastic bag that they be correctly identified by a label which will not wash off or fade when bleach is sprayed all over them. After the last growing bottle is stoppered and with the growing containers still horizontal, they are removed and put to one side before transfer to their ultimate growing position. If more seed sowing or replating is to be done, the tools are resanitised either with heat or with more bleach - excess bleach solution is run out of the bag into the sink, the bag is resprayed and the process is repeated. I have not used one bag for more than two consecutive sowing sessions and actually the plastic bags are probably the cheapest piece of equipment.

WHERE TO GROW
Your bottles of seed can be placed for germination where your tillandsias are already growing or they can be placed on a shaded windowsill. I put them under fluorescent light on a six hour light / six hour dark cycle in the belief that this speeds up the rate of growth. Certainly, six hours of light is enough for satisfactory photosynthesis. I have not conducted a controlled trial comparing a twelve hour dark / light cycle. The six hour cycle is regulated by a simple timer plug obtainable for about $20.00 at the local supermarket. I use ordinary fluorescent tubes over what was an old fish tank but better results may be achieved by special Grolux tubes and any covered area which can be kept dark during the day would be suitable for short cycle growth. Within a week you will know if your sterilisation procedures have been successful and within two weeks, small green blobs will appear at the end of the seed attached to the coma hair. Rarely is 100% germination achieved and with Seed Bank seed I am very happy if any germination occurs. Obviously, the fresher the seed the more likelihood there is of good germination.

Once placed in a suitable growing area your bottles can be virtually forgotten although it is hard to resist the urge to peer at them a couple of times a day, at least for the first few weeks. The growth rate of Tillandsioid seedlings varies considerably and the most rapid growth I have achieved has been with T. caput-medusae and T. streptophylla. Within twelve months the seedlings are approximately 1cm in diameter and even before this stage they would benefit from reflasking. Similar preparations are made and more bottles of sterilised growing medium are necessary. These, along with the bottle containing your small plants and your instruments are placed inside the plastic bag, sprayed thoroughly with bleach and left for a short period. With bottles resting horizontally, lids are removed and using angled forceps or a wire hook, plants and clumps of plants are removed from the germination flask and placed individually, with a space of about 1 cm between plants, onto but not necessarily into the new growing medium. Don't worry if old seed or hair is also transferred. This would be sterile. Flasks are restoppered and the new flasks are returned to the growing area. Sometimes with poor germination, reflasking is not necessary and I sinply leave the few plants to grow to a suitable size where survival in the outside world can confidently be expected.

DEFLASKING:
One of the many advantages of this method is that you can choose the most suitable season and time to transfer your plants to the outside world. Lately, my time has been limited and there are a few bottles of plants growing happily under lights in which the seed was sown years ago and they have never been touched since. Their growth rate is less than ideal but they are living quite happily and will wait until it is convenient for me to attend to them. The growing on area should be slightly more humid initially than your normal tillandsia growing area.

To begin with, I soaked the plants in systemic fungicide solution - a combination of Benlate and Fungorid, but lately this step has been omitted, again without apparently causing any fungal problems. The grey leafed tillandsias are grown on old palings or cork slabs. Simply wind fishing line or nylon knitting ribbon around the paling firmly. After washing the agar jelly off the plantlets under running water, slip them under the fishing line or allow their roots to be held by this support. Vrieseas, guzmanias and green leafed tillandsias are usually transferred to community pots with sphagnum moss on the surface. For the first week or so humidity is maintained by the method Olwen Ferris first showed me Cut the top off a suitably sized plastic soft drink bottle and invert this over the pot. After the initial watering no further watering is carried out until the cover is removed after three or four weeks. Further growth proceeds in the usual growing environment of these plants maintaining a fairly high humidity in the heat of Summer. This also applies to the palings on which silver-leaved tillandsia plants are grown although it amazes me how quickly the green immature leaves acquire a silvery covering of trichomes. This can occur within a week and often within a month, small green tipped rootlets are attaching the plants to their sub-strate.

The advantages of this method are: -

  1. TIME - The initial preparation of media, soaking of seeds, disinfection of seeds, setting up the plastic bag and seed transfer would probably take two to three hours for enough medium to grow twenty separate bottles. After this, you can virtually forget about them. No worries about maintaining humidity by watering two or three times a day at the height of Summer or worrying about too much cold or damp in the middle of winter. The lights appear to provide sufficient warmth for growth during the coldest winter months.

  2. CONVENIENCE - Apart from attempting to sow seed as soon after harvest as possible, everything can be done at your own convenience and one is not tied to one's growing area for fear of atmospheric change. For viable seed there is a very small mortality rate from germination to deflasking compared to the possible mortality rate with other methods of culture.

  3. OTHER SEED - This method works equally well with both green-leaved and silver-leaved tillandsia species. In fact it works extremely well with Aechmea, Billbergia, Pitcairnia and Quesnelia seed in my experience. The plant growth in these species is extremely rapid and plants hit the top of the bottle in a couple of months and on deflasking into community pots, appear to have an excellent rate of survival in my growing conditions.

DISADVANTAGES:

    1. Initial cost - I feel that the investment in a pressure cooker is well worthwhile and your wife may also appreciate the opportunity of having an alternative cooking method.
    2. Plastic bags are cheap. A small cost is involved in obtaining the steel wire and glass rod and a gas cylinder with a flame to melt glass to make handles.
    3. The latest quote from BACTO for sufficient orchid mother flask medium to make up one litre of solution was $12.00 but remember this will probably make up at least twenty bottles ($84 for 500gm).

  1. Finding suitable glass containers - I obtained mine in the hospital system, but small pure fruit juice bottles would be equally suitable and test - tubes are reasonably cheap. All glass containers are reusable.

  2. Possible sensitivity to bleach. I frequently develop a rash on my forearm where the rubber band rubs with movement. This lasts about a week and probably should teach me to wear a glove when carrying out this procedure.

  3. WHAT TO DO WITH ALL THE BEAUTIFUL PLANTS which are about to flower four years after you have sown the seed? There is no easy answer to this minor problem.


FURTHER READING.

  1. Home Orchid Growing, 1970. Rebecca Tyson Northen. ch.9, Growing and sowing your own seed.

  2. The Biology of the Bromeliads, 1980. David H.BENZING. Ch.16, Comments and techniques for the bromeliad grower.

  3. Proceedings Bromeliads III Conference, 1985. Peter PAROZ p35 Seed Raising - Xerophytic Epiphytes. Fay SIBLEY p39 Seed Raising - Mesophytes.

  4. Journal Brom. Soc., 1990. Vol 40 No 1. Mark A.DIMMITT. Growing atmospheric Tillandsias from seed.

  5. American Orchid Society Bulletin, 1990. Vol 59 No 10. Marilyn H.S.LIGHT. Doing It! Sterile Technique, Flasking, the Works.

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