Micropropagate banana? Whatever for? My plants produce lots of pups why bother? A good question, for which there are several answers:

1) Micropropagation (MP) allows for more rapid division of desirable plants. One meristem can yield dozens, to hundreds to thousands of clones in months. Of course, one can limit the numbers of new plants should one choose - but MP is the fastest, most efficient method for multiplying your banana plants.

2) MP eliminates a number of diseases (fungal, bacterial and parasitic) and reduces the incidence of several others (viral disease). This allows for the transfer of plants with little worry of contaminating ones growing space with banana pathogens.

3) Space - with MP one can ship multiple shoots ina small container, each of which will rapidly grow to a new plant, or can be divided by the recipient denpending on his/her need.

4) Storage - one can maintain more varieties on a shelf than one can on an acre of land!

6) There are a number of studies that show that TC propagated plants reach maturity faster and fruit earlier. This is for commercial banana farms. I have seen is said in a number of online places that using banana corms result in faster growing plants. It would appear that this may well be the case in more marginal banana growing environments, like most of the US. It is my feeling that in most of the continental US, that comparing TC plantlets to corms is an apples to oranges proposition.

5) It is a fun and exciting challenge!

On this webpage I will describe the materials you will need, where you can get them, and of course, how to use them to micropropagate (MP) your own banana plants at home. I was discouraged from banana MP early on as professional tissue culture labs were very reluctant to sell sterile materials, or to divulge �secrets�. Fortunately there is abundant literature out there and some academics are very willing to share their expertise. I would alss like to put in a plug for Carol Stiff�s Kitchen culture kits group at yeahoogroups. Nice bunch of folks all interested in �at home� MP. Carol is an experienced tissue culturist and is very helpful to the neophytes. Her kits are top notch as is her CD.

Apical Meristem: This is a region of undifferentiated tissue that dominates the plant and suppresses the development of secondary meristems. In bananas this is the growth point at the base of the pseudostem from which all leaf primordial arise. We will be using a growth regulator, the cytokinine BAP to break this suppression and allow the development of multiple meristems.
4 basic stages of MP:
Initiation/establishment
Multiplication
Rooting
Acclimatization/hardening off
Growth Regulators: Used to be called hormones, but the term growth regulator is considered more accurate. These are substances that are either natural are internally-secreted chemicals or synthetic analogs in plants that are used for regulating the plants' growth Cytokine: The most important cytokine for our purposes is BAP or 6-benzylaminopurine. This substance reduced apical meristem dominance and allows for other meristems to grow and produce new plants.
Auxin: while there are some banana media that make use of auxins, they are not strictly necessary for banana MP
Media: Sterile material that will support all necessary physiologic functions of the plant and deliver necessary growth regulators to effect specific changes

This is miniature controlled vegetative propagation, in essence we will be miniaturize Bananas, and then accelerating the normal vegetative reproduction of the plants. Banana micropropagation is not strictly tissue culture but is best termed micropropagation or �shoot tip culture�. With bananas the stages of initiation and multiplication are combined and the Media is identical.

You will need:

Sterile Transfer Environment
Culture vessels
Forceps
pressure cooker
Scale, electronic (no mandatory)
pH meter, pH paper (not mandatory)
Bleach, household variety
Isopropyl alcohol, 70%
Clean, preferably distilled water
Scalpels or razor blades
Media

Sterile Transfer Environment: What does this mean? This is basically a covered area that will exclude bacteria and fungal spores from entering your culture vessels. This can range in complexity from a cardboard box to a laminar flow hood. Carol Stiff's Page has a very nice description of different transfer case designs. Dr. Henry Kuska has created a small, but effective HEPA filtered laminar flow hood from house hold materials.

Does the average hobbyist need a fancy Laminar Flow Hood (LFH)? Nope. I do all of my transfers inside a plastic sweater box. The box is wiped out with isopropyl alcohol and the opening is covered with plastic wrap. Some folks will disinfect with 10% bleach, but the fumes can be quite irritating. I let this sit for 20+ minutes to give the alcohol a chance to kill as many bacteria as possible. The lowest 4 inches of the plastic wrap is simply lifted up and a space to pass my hands is made that runs the entire length of the space. Work slowly and carefully and contamination should be minimal.

Culture Vessels: There are a number of different culture vessels that will work for banana MP. I use Baby food jars with Magenta B caps. These are autoclavable plastic caps that are designed to fit standard baby food jars. You can buy these caps from almost every TC supplier, or if you are using a Kitchen Culture Kit (KCK) , you will get them with your kit. Baby food Jars can be purchased from many TC suppliers, but a few phone calls to local day care centers will have you well supplied in no time. If you can�t find Magenta B caps, the Phytotechnology version, the Phytocap is an excellent choice.

I have also used Deli cups and Mason Jars as culture vessels. These are vented with a small 1/32 inch hole drilled in the side or lid and covered inside and out with 3M Nexcare round bandages. These allow for gas exchange but minimized the movement of contaminants. There are a lot of different vessels available and as long as they can provide some gas exchange while remaining sterile, they will be adequate for banana MP. Check out any of the TC suppliers and see what works for you.

Finally, it never hurts to add a little insurance when protecting your cultures from the odd spore adrift in the air. IF you don�t have a clean room to store you cultures, and what hobbyist does, you should seal the vessels using either ,A hreaf=�http://www.phytotechlab.com/detail.aspx?ID=737�>parafilm or Seran wrap. I just cut a roll of Seran wrap into 1 inch wide strips and wind it around the closure. Cheap, effective and very easy to remove.

Forceps: I like 6-10 inch stainless steel forceps for manipulating cultures. They should be long enough to keep you hands away from your plant material, but not so long that you have trouble manipulating them.

Pressure Cooker: In professional labs, media and instruments are sterilized in an autoclave. This is essentially a giant pressure cooker. They are expensive and unwieldy - fortunately for us, a standard pressure cooker will work quite well. There are a myriad number of models out there. Some are enormous and others are quite small - with space for only a few jars of media. Shop around and get something that can handle 8-10 jars at a time, or you will be forever sterilizing media and never get to your plants!

The pressure cooker uses steam to heat the contents, and because everything is at an elevated pressure, the temperature is higher than the 212 degrees F that water will boil at. (Boiling is not sufficient to sterilize media - some bugs can survive it!).

There are some home tissue culture enthusiasts that advocate the use of a microwave for sterilization. Personally I find that this is less reliable than the pressure cooker, and if it boils over - quite messy. If you would prefer to use this method please see Carol's page for directions, or consult your KCK manual - there is a very nice explanation there.

Scale, electronic: I used to think this is essential. If you do invest in one, the smaller the units of measurement, the better. You will want one that goes down to 0.1 grams if at all possible. These can be had from chemical supply companies, TC suppliers, police auctions (some folks use these to measure small quantities of illegal substances) and office supply companies. You can even buy some nice scales from the ads in pet bird magazines. Do not use this scale to measure foods - keep it separate for your TC only.

Now you can get some very nice, pre-measured media directly from phytotechnology Labs. Having that negates the need for a scale.

pH meter, pH paper: Plants can be persnickety about the pH of the media they are being placed in - therefore it is important to be able to measure the pH of the media you are making up. There are several options here. The best would be an electronic meter. These range in price from 40-500 dollars. For our purposes the lower end of this scale is adequate. The following meter has been suggested to me and has several fans out there in Home TC-land. Edvotek Checker 1 pH meter (#577) and lists for $39.00, phone 1-800-EDVOTEK, or FAX 1-301-340-0582. If you are making allot of media, then $39.00 is a small price to paw for consistency and quality control.

Now if you buy some of that prepackaged stuff, and use distilled water, there is no need for the pH meter. Phytotech to the rescue.

Other items on the list: I think these are self explainatory: Bleach, household variety
Isopropyl alcohol, 70%
Clean, preferably distilled water
Scalpels or razor blades

A number of recipes have been published for Musa MP. I will describe the one that I use. This is Linsmaer and Skoog media with BAP as the growth regulator. I firmly believe in keeping things simple. To that end I will suggest that you simply buy Musa (banana) multiplication medium from Phytotech as you literally just add water, dissolve, portion out and autoclave. Buy the pre-measured one liter bottles of powder. They are a little more expensive, but you would have to make a lot of media to make up for the price of a scale. If you plan on turning this into a business, it will be cheaper to make your won media from the components, but if you are using less than say 50 liters of media per year, there is no real savings to be had. After all, time is money, and the fun part of MP is not media preparation. Once you have multiplies your bananas you will be looking for a rooting medium. This is simply the same media without the Cytokines.

But if you heave your heart set on making your own media, consider the following recipe, it is very simple and very effective.

One liter of this media contains:
4.3 gm Murashige and Skoog salts
100 mg inositol
0.4 mg thiamine
5 mg BAP
1 ml PPM
2-2.5 gm Gellright
pH=5.8

There are a number of other materials that researchers have added to the above, but to use them will net little benefit for the added effort and expense for the hobbyist.

I have dissolved my BAP in dimethylsulfoxide (DMSO). This material is not generally available to the public and can be hard to get. The concentration of BAP in DMSO that I use is 25 mg/ml. It can also be dissolved in 1M NaOH and then in water. If you use water, the concentration that you will use will be 1 mg/ml. The aqueous form is very alkaline and you will need to watch your media pH. You can also buy this over the internet as an aqueous solution 6-benzylaminopurine solution (1.0 mg/mL)

It is important to create a situation where the tissues are not submerged in the media as they can drown. What is needed is something that stiffens the media yet allows for the various components to diffuse to the tissues and allows waste productes to diffuse away from the tissues. There are several gelling agents available for use in plant TC. If you are doing research you will want to use one of the TC grade materials. The reality is that your banana plants will not care much and you can use everything from food grade agar, to Gellright, to sand or cotton balls. There is some research that suggests that gellan gum (also know as Gellright) is superior for banana work. I prefer it because it is less expensive than agar and the gel if forms is crystal clear. This allows for rapid detection of contamination.

1) Measure out 1 liter of distilled water. (If you do not have an accurate volumetric flask don't worry - 1 liter of water weighs 1 kg - use your scale!).

2) To this add the contents of a packet of MS salts. If you use MS media with the thiamin/inositol already added you can skip the next step.

3) Add 1 cc of the vitamin (thiamin/inositol) mixture to the media.

4) Add 0.2 ml of the 25 mg/ml BAP in DMSO, this will deliver 5 mg BAP to the media.

5) Add one ml of PPM

6) Add several drops of food coloring to identify the formula (I use several different media formulations and the color coding helps to differentiate them - thanks for the tip Carol!!)

7) I measure the pH of this mixture and adjust it to 5.8 using vinegar if the pH is too high or a crushed antacid tablet if the pH is too low. Use only tiny amounts and mix well with each addition - a little goes a long way!

8) Measure out 250 ml of media into a heat resistant container.

9) To this add 0.5-0.6 gm of gelright and swirl to disperse the powder.

10) Microwave this until it just begins to bubble and the pour approximately 20-25 cc of the media into each baby food jar.

11) Cap each jar with a magenta B cap and pressure cook it for 15 minutes.

12) Remove the bottles to cool on a baking rack before use. If the media is not used straight off, refrigerate it for up to one month.

Identifying and disinfecting of the explant is the first, most important step in establishing any plant in TC. We are lucky in that the meristem of the banana is very clean - perhaps even sterile. What we will be doing is reducing that amount of surrounding tissue to allow for easy access to the meristem, surface sterilizing this and then trimming down to the size we want. This has the added benefit of hopefully cutting away any tissue that is not perfectly sterile.

Harvest a sucker that is at least 3 inches in diameter. Suckers are the preferred material as they are in an actively growing phase and can be taken without destroying the mother plant. For those who do not have any suckers on their banana plants (just wait - they'll be there!) you can use the mother plant.

Kitchen Culture Kit (KCK)

Lydiane (Ann) Kyte

Plant Tissue Culture for Home Gardeners

Cloning Plants by Tissue Culture by Michael H. Renfroe

Tissue culture in the Home Kitchen by Rick Walker

Listservers

Kitchen Culture Kit (KCK) This is a place for great information, the homepage of the Home Tissue Culture Listserver, and a fantastic source of information. The KCK is an excellent way to get started in the hobby and comes with a fantastic handbook full of good info. Check it out!

Phytotechnology Laboratories One of the best TC supply companies in the business. The service is excellent and the products are superb. Basically I order everything from this company and have been nothing but happy with them.

This is a great forum with banana enthusiasts from around the world � join up and talk bananas!