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Home Plant Tissue Culture Made Easy

IMG_6334More and more plant tissue cultured aquarium plants are coming available.  They’re becoming affordable too and we can see them in many shops regularly now.  Most plants already in tissue culture can easily be put into multiplication cultures so that you too can grow masses of plants easily.  They even store well in a library type collection in a suspended manner for months if stored properly.  What’s keeping you from trying plant tissue culture?  Here’s how it can be done with minimal specialty tools and gear.

The sterile media in the cultures is the most important aspect of home plant tissue culture.  Media that suits the purpose of each stage is put into a jar and sterilized in an autoclave.  An autoclave can be substituted by using a pressure food cooker or a microwave.  A microwave cannot have metal inserted in it so you need plastic containers.  Not a big deal.  For the pressure cooker any type of small jar with a lid can be used.

The first step is to make your media to grow your plants in.  This can be ordered from a supply house like PhytoPlant Technology as Murashige and Skoog media (MS) or can be made at home using a diluted fertilizer.  You will need sucrose in the form of white table sugar and most plants will benefit from having a gelling agent like agar to make the media support the plants upright.  Agar is available in most Asian markets in a small packet.

Cells of a plant are totipotent, meaning that they can become a root, a leaf, or whatever external influences guide it.  We can manipulate that with plant growth regulators.  These chemicals are readily available inexpensively on sites like Ebay and PhytoTech Technology.  For most of the aquatic plants you will need to acquire BAP also referred to as BA, 6-Benzylaminopurine, or benzyl adenine.  It is a cytokinin that promotes accelerated cell growth.

You will also need some small tweezers or forceps to flask and deflask cultured plants with.  They will need to be long enough to reach the bottom of your vessels without your touching the lip of the jars. A pair of small medical type all metal scissors and a scalpel will come in handy but can be worked around if you can manipulate the tweezers if you are just replating established cultures into new multiplication media jars.

You will need a scale that measures milligrams somewhat accurately.  It will be used to measure your fertilizers and plant growth regulators in order to get accurate amounts into various solutions and media mixes.  You’ll need to measure the dry components.  You can work around this using the smidgeon measurements but a scale makes life much easier.  Plus having a scale comes in handy for dosing your aquarium with dry fertilizers.  Inexpensive scales can be bought off Ebay for 5 or 6 dollars.  You get what you pay for but I grew ten thousand plants with a cheap Ebay scale.

IMG_6335All the media jars need to be sterilized in order to be productive.  The absolute best way to sterilize your vessels is to use a pressure canner for home use.  The pressure canner increases the temperature of water boiling by adding pressure.  The higher the pressure the higher the temperatures created inside.  Presto makes a 23 quart model that is perfect for the small home tissue culture practice.

The second option for sterilizing your vessels is a microwave.  The containers are placed inside the microwave and closely monitored for boil over.  The containers will want to boil over so you have to monitor them closely by turning off the microwave and restarting it frequently.  Some models have an option to reduce the percent of heat and this can be used to avoid having to turn the microwave on and off so many times in a session.

Once the media has been sterilized and left to cool it is ready to use.  Whether it is initiation or multiplication media care should be taken to avoid extreme changes in temperature.  Let the containers cool naturally int he microwave or the pressure cooker.  Fast cooling can cause the intake of contamination when the hot media and air inside the container shrinks form the difference in temperature.

You will need some sort of clean hood to insert your plant material into sterilize vessels.  One of the easiest to obtain is a rubbermaid container or a 20 gallon high glass aquarium.  They are both easy to clean.  Glass is the most inert and repeated exposure to sterilants like bleach and rubbing alcohol don’t cause it to break down like acrylic will, or even plastics for that matter.

Bleach is a cheap and easy to obtain sterilant and it works very well.  A 20 percent bleach solution will work very well for sterilizing your hood and outside of your vessels.  A spray bottle atomizer works best for applying the bleach to the surfaces.  The finer the atomization the better.  It also reduces the amount you have to spray which saves on mess and smell.  There are other ways to sterilize but bleach is the easiest and cheapest.

Putting plant material divided up from an existing culture is called replating.  Replating your cultures is how you multiply in general.  There are other reasons to replate but for this example we are going to use it for multiplication vessels.  The plant tissue culture you obtained is simply divided up into a group of material and each one put into a new jar.  As an example let us assume that you have a TC tub of Cryptocoryne Pink Flamingo.  This group can be divided into 10 new vessels carefully and in about a month you will have ten new vessels of Crypto Pink Flamingo on their way to becoming 100 new vessels when divided.

The existing culture along with your tools and new jars are put under the hood after it has been sprayed with bleach.  With latex or nitrile gloves spray the inside of the hood liberally with a 20 percent bleach solution.  Then add each vessel you are going to be working on one at a time being careful to mist each and every single item going under the hood.  Wear an N95 type face mask to avoid breathing into the protected area of the hood.

With sterilized tools, which are sprayed with bleach and rinsed in sterile water (done when sterilizing media) separate the existing culture carefully.  Avoid touching anything not impossible not to touch.  Avoid touching the rims of the vessels.  Do not let your tools touch the bottom or sides of the hood.  Be as careful as possible to not contaminate your cultures.  Remember the more careful you are the more successful you will be.  Work with one jar at a time carefully.  The mass of plants you took out should be carefully divided and placed into a jar.  Each jar should be carefully opened, avoiding touching the inside lid or jar area.  Place the plant material into the bottom of the media and close up the jar.  Place them out of the way in the back of the hood.  Once all the jars are done you can remove them, but if you take your hands out from under the hood make sure to spray them with bleach before starting back until the hood.

When the jars are taken out of the hood area, wipe them down.  They should be stored in subdued lighting with some way to protect them from dust.  Using rubbermaid container with lids greatly reduces any dust that may fall on them and reduces the risk of contamination.  Label each vessel with the media you used and any plant growth regulators you added.

In a month or two you will have hopefully thriving cultures of tissue cultured plants.  These can be taken out and grown or they can be divided and multiplied again.  Since cultures can be kept in their media when the nutrients run out they can be kept in a sort of suspended state.  Make sure to monitor them and be ready to replate them if they begin to look weak or decline.

Cultured plants are easy to ship and make for trading and building a plant library pretty easy.  Large amounts of plants can be kept in a small space.  Tissue cultured plants are free of snails and other pests and can be acclimated to emersed growth or aquarium growth rather readily.  Once you get the hang of it you’ll be initiating your own cultures from new specimens.

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