(actually, the last point is not quite true. Scott Hyndman, marva@nebula.ispace.com, informs me that Clonal integrity is influenced by stress factors, genotype, culture age, cutting and transfering technique, and numerous other factors still being investigated in the voluminous scientific literature on just this fascinating aspect of plant tissue culture alone.)
Totipotency:
certain cells have the capacity, when isolated and properly
grown, to regenerate a whole plant. This is nothing strange or
unusual. The picture shown here shows how the common "spider plant"
is capable of starting new growth at the end of each shoot.
Meristem:
The region in a growing plant where the cells are rapidly dividing.
The picture shows how the meristem can be isolated from a
strawberry shoot to be grown out in sterile tissue culture.
Both these pictures are reproduced from "Plants from Test Tubes" by Lydian Kyte. This book is highly recommended as a reference for beginners, and a full bibliographic reference is given later in this talk.
More accurately, this should be called a talk on "in-vitro" propagation of carnivorous plants. "In-vitro" means "in glass", or under sterile conditions.
It is very difficult (or sometimes nearly impossible as in Nepenthes) to properly sterilize meristem tissue from many CP. Some of them have symbiotic fungii living within their cells. This stuff usually breaks out and overruns the culture when meristemming is attempted from non-sterile material.
For this reason, the most reliable way to start a CP cell line is from seeds.
Once you have a sterile culture going, then you can multiply the tissue with hormones, and re-divide ad-nauseum. For some CP, notably Pinguicula and Sarracenia, it is possible to do meristem techniques with normally grown plant material. I won't cover the dissecting procedure here, but the sterile technique, media preparation, etc., are identical to the in-vitro procedures shown here.
Toby Marsden defines the three classical stages of hormone-assisted TC as:
For home tissue culture, hormones are not necessary. Most plantlets such as Pinguicula already grow much faster in TC than in soil. Unless you need extremely fast multiplication for commercial purposes, or are experimenting with meristem propagation of very difficult species (eg: Nepenthes ), it is unlikely that you will need to use hormones or cytokinins. Many of these chemicals are dangerous (mutagenic or carcinogenic), and are not really appropriate to be using in the kitchen.
This photo shows a package of Murashige and Skoog (MS) Tissue Culture media as sold by SIGMA chemical company. Each packet provides enough chemicals to prepare 1L of media. To use this media for CP, it generally needs to be diluted in strength. I usually use 1/2 strength for most non-critical plants. The four bottles shown each contain 1/4 of the SIGMA packet, built back up to proper sucrose and agar concentration. For most CP, 20-30g/L sucrose and 6g/L agar is appropriate.
Each bottle then conveniently contains the proper chemicals for a 500mL batch of media.
The next slide covers the functions of the major nutrients in TC media.
punching an air hole in the lid
using foil as a contaminant barrier
Baby-food jars make excellent and inexpensive growing chambers. For the beginner, a hole may be made in the lid with a nail and hammer. To prevent contamination, the lid is kept wrapped with aluminum foil. This allows slight breathing of the media while providing a baffle to exclude bacteria and spores.
The more advanced TC enthusiast may wish to purchase plastic caps from a commercial source such as SIGMA chemical company. I use the "MAGENTA B-cap". They are sterilizable, provide baffles, and are transparent to light. This makes it easy to grow the cultures with overhead illumination. These caps are designed to be used with standard baby-food jars.
Tissue culture does not need to be a "high-tech" affair. Many non-fussy plants can be easily grown on a cookbook "kitchen-style" media. Here is a simple one, adapted from Kyte, to try for Pinguicula and Drosera. There is a lot of room for experimentation, and this formula can certainly be improved with some trial and error.
components of a simple homemade media
For variation, try substituting coconut milk in place of some of the water. Common concentrations used in the literature are 100 to 200 ml of Coconut milk per liter of media.
Inositol is a common human food supplement and can be found at health-food stores in gel-caps. Agar is sometimes available at the same stores in bulk bins. Another place to look for agar is at Oriental food specialty stores. Agar is commonly used in Asian cooking as a gelling agent for desserts. Get the whitest and purest, unflavored variety that you can find.
You may have to experiment with the concentration when you use non-standardized agars. Your goal is to achieve the minimum concentration of agar that still reliably forms a gel. This will produce a media that offers minimal resistance to root growth.
Note: This recipe was originally designed for general purpose use. Most CP require a more dilute nutrient concentration than non-CP, so you might try reducing proportions of stock solution and sugar until best results are obtained.
This recipe is adapted from the highly recommended book "Plants from Test Tubes" by Lydian Kyte, published by Timber Press (see references at the end of this document for ordering information).
This is the Pinguicula Media recommended by Bill Carroll in the Carnivorous Plant Newsletter, v11 n4, December, 1982 pp. 93-96. As you can see, you probably don't want to attempt this one without an accurate balance and a well-stocked supply of chemicals.
For shoot multiplication use Kinetin or 2iP in range of 0.5 to 2.0 mg/liter. Auxins for rooting were IBA or NAA in range of 0.1 to 1.0 mg/liter.
Bring media to boil while stirring constantly, dispense into test tubes or other containers and steam sterilize for 15 minutes at 15PSI (120C or 250F).
Whatever formula you use, you need to mix it up according to the manufacturers recommendations, or according to your formula. I usually heat the water almost to boiling before adding the Agar with vigorous stirring. When fully dissolved, you can dispense 1-1.5 cm of media into each Baby-food jar. Be careful not to get any media on the rim or sides of the jars as this will later provide a path for contamination. Replace the (vented!) lids on the jars and stack them into the pressure cooker for sterilizing.
Loading a simple pressure cooker
Make sure and use a trivet to hold your jars up off the bottom of the cooker. You want to steam-sterilize them, not boil them!
This small, skillet-sized pressure cooker came from the Whole Earth Store for about $200. It's a fairly pricey, high-end stainless-steel model for gourmet cooking. I included it here as an example of "making-do" with what is available. This canner is just big enough to barely fit 7 short baby food jars.
Loading a bigger "Home Canner" pressure cooker
This is a bit more industrial sort of canner, made by "American Aluminum Company". It's very nice for sterilizing big batches of media, tools, and for sterilizing rinse water.
I purchased this one mail-order from Mellinger's [see refs. for address], but the same canner is also available locally at Orchard Supply and Ace hardware. Smaller units from the same company can be had for around $70 or so.
Although Microwaves have been used for sterilization (see bibliography), the results have been spotty. The dividing line between achieving sterilization and flash-overboiling the media is extremely fine. Even under the best circumstances, the incidence of contamination is much higher with a microwave than with a pressure cooker. If your environment has a high concentration of heat-resistant spores, then microwave sterilization will probably be unusable.
With a pressure cooker you can assume, with near certainty, that your media is absolutely sterile. Then you can focus your attention on reducing contamination during seed sowing, cell transfer, etc. I highly recommend using a pressure cooker for any serious TC work.
Whichever type of pressure cooker you choose, they all should be set for 15 PSI/250F, and run for 15-20 minutes.
I usually leave the check valve open until it starts to discharge a good quantity of steam. At that point, you are assured that the vessel is well-filled with live steam. You can then close the valve, lower the heat, and start the timer once you've come up to pressure.
Never leave a cooker unattended. Please read all the safety directions for your cooker before starting!
Make sure and let the cooker come to room temperature before opening the vessel. If you don't, then your media is likely to burst into a boil and foam all over the place. I usually leave the cooker overnight before opening it. This also has the advantage that the agar will be fully gelled, and there will be no problem with spilling the media when removing the jars.
After things have completely cooled, you can safely open the cooker. There may be a slight internal vacuum which could suck in contaminated room air. It is suggested that the cooker checkvalve be wrapped in paper toweling that has been soaked with isopropyl alcohol. Carefully release the internal vacuum by opening the check valve. The room air will then be filtered by the paper toweling.
labelled seeds on filter paper
folding paper and securing with plastic clip
I sterilize seeds in a little folded packet of filter paper by soaking for 5 minutes in isopropyl alcohol (frequently shaken or stirred), 2-4 minutes of 1/10 Clorox solution, and 1-2 minutes of 3% H2O2 as a final rinse. I leave the residual peroxide on the seed as a further infection prevention measure. Some workers prefer to rinse all traces of chemicals off the seeds with pre-sterilized water.
It can help to add a drop of detergent to the bleach solution to allow better wetting of the oily seed coat.
In private correspondence, Jan Schlauer has recommended judging the bleach timing by looking at the color of the seed coat. When you have just noticed a change in color (from black to brown, or from brown to straw-colored), this is about the right time to stop the chlorine disinfecting step.
It is always a delicate juggling of trying to kill the contaminants without killing the seed. For the best chance of success, you may want to divide your seed into several batches. Process one batch for 1 minute, the next for 2, and the last for 4 minutes. Sow them in seperate flasks and keep good records. This will help you to perfect your judgment and technique.
A commercial laminar-flow hood
This is another line drawing from Lydian Kyte's book: "Plants from Test Tubes". A commercial hood like this can cost perhaps $1500.00. You can buy the filter and fan units separately to make your own for much cheaper if you are handy with working in plexiglass.
In a laminar-flow hood, the incoming air is filtered by a High-Efficiency-Particulite Air Filter (HEPA) and flows smoothly over the work area. The HEPA filter is fine enough to completely remove mold spores and bacteria from the air stream.
John Laroche has written a simple "howto" describing a how to build a Glove box, Laminar Flow hood and a culture rotator.
For the hobbyest, good results can be obtained with the much simpler system illustrated below. This is an adaptation of the "glove box" type of transfer chamber.
This is a 40 Gallon Aquarium, turned on its side, and covered with a curtain of plastic sheeting. I use the overhead fluorescent fixture for lighting. The clear section of glass in front of the light fixture is where to look for a clear view.
Prior to using the chamber, you should swab down the inside of the box with a paper towel moistened with Isopropyl. Be very careful to let the fumes dissipate prior to lighting your lamp! In this regard, Isopropyl (rubbing alcohol) is much safer than, say, Lysol Spray, which contains Ethanol (grain alcohol) and is much more flammable.
To further safeguard against contamination, you may want to buy a home air filtering unit. The Holmes company makes a unit with a true HEPA filter, for about $70.00. I usually leave the unit running for a day or so prior to doing sterile transfer work. This greatly reduces much of the air-borne dust in the home environment.
This same chamber serves nicely as a growing area.
cleaning hands prior to beginning work
I clean my hands with soap and water, and rub them down with isopropyl. Plastic surgical gloves can also be worn, if desired. A short sleeve shirt is recommended to avoid carrying particles in with the fabric.
A good watch is useful for timing the sterilization steps. (Make sure you think through your technique... I've sometimes gotten involved in sowing seeds, and left others to soak for 20 minutes in bleach... this is not recommended!)
Another useful tool is a pair of tweezers. These should be fairly long so that you can manipulate the material without getting your hands too close to the agar. An 8" piece of thin, stiff wire with tip fashioned into a 1/8" loop is helpful for sowing seeds.
A pair of forceps and a razor blade or sharp knife for dividing clumps of plants may also be needed.
alcohol lamp for sterilizing tools
A camping burner or at least a candle will be needed in order to sterilize the wire loop during the sowing procedure. I use a lab-style alcohol lamp. This type of lamp has a broad base to prevent tipping. Be careful!
Also, note that Isopropyl alcohol does not burn well at all. You will need to buy denatured methyl alcohol for your lamp. This fuel burns cleanly and leaves no residue on the tools.
sterilizing innoculating loop in flame
I usually dip my tools in isopropyl and then "flame" them off in the alcohol lamp. This helps to sterilize both the shaft and tip of the tool. After "flaming" off the isopropyl, I then heat the tip of the loop until it glow red. It can then be inserted in the gel while still hot to cool it down. This technique serves two purposes: 1) it keeps the tip hot to protect from contaminants, and 2) it picks up a little gel on the tip to make it "sticky". This bit of stickyness will help to pick up the seeds in the next step.
Notice the MAGENTA B-CAP held in the curl of the right hand. This is a helpful sterile lab technique that is worth practicing. Here is the procedure:
As usual, there is a trade-off to be made here. The more seeds you sow, the better your chance of having a successful germination - BUT - also the higher chance of contamination. For easily sterilized glossy seeds such as Dionaea , I usually sow up to 20 seeds. For tougher seeds, like Nepenthes , you might be better advised to only sow 3 or 4.
Pinguicula heterophylla in culture
plants in-vitro and also planted out
I have kept my cultures under coolish home temperatures (60-75 degrees F), 12 inches below a two-tube 40W fluorescent light fixture. I keep the culture jars inside the same glass aquarium that I used as the sowing chamber. This helps to reduce contamination from air-borne dust.
After your plantlets have reached the size of a pea, you have a choice of further multiplying them in-vitro, or transferring them out to grow in regular soil. If you choose to multiply your plants further, you initiate the process by simply cutting up your sterile material and moving it into new media. At each stage, you may be able to increase the number of flasks by over 10 fold.
Of course, all dissection work must be done under sterile conditions. A laminar-flow hood is really handy here, as the plant material will be exposed to possible contaminants for an extended period of time.
If you are skillfull and quick, it can still be done with minimal equipment.
The key to sucessfully transferring your in-vitro plantlets into soil is to be very fastidious about washing off all the TC media from the roots.
I usually put the plantlet under running, tepid water, and use the force of the water to thoroughly dissolve off all the old media. If this is not done, then molds will inevitably take hold and overpower your plants.
After planting out, treat the plants the same as they were treated while still in-vitro. A humidity tent made with a zip-lock bag will help the plants acclimatize. Let them stay sealed for a week or so. You can then gradually open up the bag over the course of another week to get the plants used to lower humidity. Once they are "hardened off" properly, you can treat them as any other soil-grown plant.
Please excuse the technical format of this list. It is organized by family and genus. Much of the info here is due to Jan Schlauer, Andreas Wistuba, John Laroche and others on the CP listserv group. Many thanks to these intrepid experimentors!
In cases where there is no formula listed, you might try using one for a related genus in the same family. Other than that, you are probably exploring new territory. Please keep good records and let us know what you find out!
*= You may add 37,26mg/l Na2EDTA and 27,8 mg/l FeSO4 x 7H2O. Andreas Wistuba recommends adding the MS-vitamins to Knudsen C medium.
Note: most media should be prepared with agar at 6g/L, and sucrose at 20g/L.
IBA is an abbreviation for indolebutyric acid
NAA is an abbreviation for naphthylacetic acid
IAA is an abbreviation for indoleacetic acid
MS is an abbreviation for Murashige and Skoog formula.
These are all growth regulators for controlling rooting, multiplication,
callus formation, etc.
Some of these sorts of chemicals are potent carcinogens and should be
treated with utmost respect. I find that for casual home TC, that most
of these are not really needed. They are used for really speeding up
growth or for getting phenomonal multiplication.
If you wish to try these out, then I recommend really delving into Lydian Kyte's book before beginning.
Commercial TC media
A scale, sugar and agar.
Plants from Test Tubes
Processing
Processing
alcohol, bleach & peroxide
soaking seeds
Simple aquarium transfer box
tools used for transfer work
picking up the seeds
sowing the seeds
showing seed placement
tranferring plantlets