Ross M. W. Bennetts

@ rossbennetts.com

March 12th, 2010

[Editors note: This article was originally published by Professor Acram Taji, but it was removed during a site restructure nearly 3 years ago. After numerous requests for people to put it back up online I have decided to publish it here because I think it is very valuable information. A snapshot of the original is available on Archive.org here]

Plant Tissue Culture for Home Gardeners

Introduction

Micropropagation is an important alternative to more conventional methods of plant propagation. It involves production of plants from very small plant parts (e.g. buds, nodes, leaf segments, root segments etc.), grown aseptically (free from any microorganism) in a container where the environment and nutrition can be controlled. The resultant plants are genetically identical to parent plants.

Whilst in a research laboratory such as that in the Agronomy and Soil Science at UNE we use many high tech equipment to achieve plant production through tissue culture, it is important to note that many home gardeners and hobbyists could substitute the high tech equipment and instruments with ordinary house hold items.

Items Needed for Home Tissue Culture:

  1. A sterile still air cabinet used to transfer plants. A fish tank on its side makes an ideal transfer cabinet. Any perspex or glass chamber with dimensions of 50 cm (length), 40 cm (height) and 40 cm (depth) could easily be made into a transfer cabinet.
  2. A pressure cooker for sterilisation of media, instruments, water, paper towelling etc.
  3. Glass jars (baby food jars are excellent) and take away food containers with lids which can withstand the heat inside a pressure cooker are ideal vessels to use.
  4. Scalpel and forceps
  5. Paper towelling or even A4 white copy paper, cut to size, can be sterilised and used for a sterile cutting surface.
  6. A spirit lamp containing ethanol for flaming the instruments (avoid using Methanol as it is toxic!).
  7. Hand held spray bottle containing 70% alcohol solution to spray the transfer chamber and other surfaces.
  8. Dilute chlorine solution e.g. 1/4 dilution of the household bleach (e.g. White King) for use in surface sterilisation of plant material.
  9. Any skin disinfectant e.g. Hibitane (obtainable from any chemist shop).
  10. Media (see below)

Media Preparation:

All the ingredients indicated below can be purchased using the super market, chemist and a health food shop.

  1. Two cups of rain water
  2. A quarter cup of sugar
  3. Fertiliser stock: 1/2 tablespoon all purpose 10:10:10 (N.P.K.) water soluble fertiliser in 1L of water: use one cup of stock for this recipe.
  4. Inositol tablets (500 mg): 1/2 tablet
  5. Vitamin tablet with thiamine: 1/2 tablet- Any multivitamin tablet may be used.
  6. Agar flakes: 4 tablespoons

This is the basic media. For preparation of multiplication and rooting media add 1/2 cup of coconut milk and 1/2 teaspoon of malt. Replacing the coconut milk with 1/2 cup of green tomato puree or 1/2 cup of freshly squeezed orange juice may produce different responses. Ensure that the pH of medium is always between 5 and 6 using narrow range pH indicator tape. Adjust pH if necessary, with acid e.g. Citric acid or base e.g bicarb soda.

Mix the ingredients in a saucepan and gently boil until the agar has dissolved, stirring continuously to stop the agar sticking and subsequently burning at the bottom of the pan. Dispense into empty glass jars, using a ladle, so that the medium is about 2 cm deep. Cover and process in a pressure cooker. Cook for 15 minutes after the pressure is reached (this will be achieved when the pressure valve starts letting steam out).

Sterilising Instruments and Other Items:

Forceps and scalpels can be sterilised by being wrapped in Alfoil and cooked in the pressure cooker for 15 minutes. These items can also be sterilised by being washed in chlorine solution or being dipped in alcohol and flamed.

Sterile water is needed for rinsing plant material and sterile paper towelling to be used as a clean surface to work on. The manipulation can be accomplished on the paper. When the operation is completed, the towel can be discarded and a new sterile surface selected from the sterile supply. The water can be sterilised in glass jars. Place paper inside a paper bag and cook them in the pressure cooker above the water level. The bag will be wet on completion of sterilisation. Transfer the bag to an oven set at 80 öC and allow the bag to dry inside the oven. Do not unwrap the papers until needed.

Sterilisation of Plant Material:

All plant material can be sterilised in diluted domestic bleach, for example White King (1/4 cup of White King + 3/4 cup of water + 1 drop of detergent- detergent acts as surfactant). Put plant pieces in a jar containing the bleach for 10-20 minutes. Agitate frequently. Discard the chlorine solution, this process will kill bacteria and fungi and sometimes some parts of the plant such as outer bud scales and softer shoots. Rinse plant pieces twice with sterile water.

Operations in the Sterile Cabinet:

Great care should be taken to ensure that your cultures are free from contamination. To achieve this do the followings:

  1. Tie back your hair, roll your sleeves up and remove your watch and other jewellery. Wash your hands thoroughly with the disinfectant solution suitable for skin application. If allergic to any disinfectant wash your hands with water and wear a pair of surgical gloves.
  2. Sterilise the inside of the cabinet by spraying with 70% alcohol and wiping dry with sterile tissue.
  3. Collect and organise all the items you will need close to or inside the cabinet.
  4. Working in the cabinet, take a sterilised piece of stem from the jar with a pair of forceps (do not touch the plant material with your hands). Also sterilise your instruments by dipping them in alcohol between each manipulation and flaming them. Small pieces, 2-3 cm long with a few leaves can be cut and transferred to agar medium. If the leaves are too large either remove them or cut them to 1/3-1/2 the size. Put one piece of the shoot into each container (it is important to have only one shoot per container at this stage so that if the shoot is contaminated it cannot spread to the others). Shut the lids of the containers. Store jars at room temperature away from direct sunlight. Leave these shoots for one month.

One of these three things will happen during this time: some of the shoots will be killed by the chlorine solution and or the toxin produced by plants themselves; some will be contaminated by fungi and bacteria; or new shoots will grow very rapidly from the axils of the stems in the uncontaminated containers. Discard the dead and infested cultures.

Shoot Multiplication:

The shoots from the previous stage may have elongated during the past four weeks. They need to be transferred to the medium containing the coconut milk (coconut milk has some growth promotory properties which makes plant segments to produce more shoots) for further multiplication.

  1. Repeat the preparation and sterilisation steps for the medium, instruments and chamber as before. Sterilise your hands as before too.
  2. Transfer the containers of shoots to one side of the cabinet and the sterilised medium at the other side.
  3. Use sterile paper towelling, scalpels and forceps as before.
  4. With a pair of forceps, remove a stem from its container, and cut on the surface of the sterile paper towelling moistened with some sterile water. It is important to do all the manipulation on a damp paper towel as these plants are very soft and can desiccate readily. The separated shoots can be transferred to the new jars. At this stage, up to five pieces of plant may be put inside each container.
  5. Store cultures as explained in the previous stage.

The multiplication stage may be repeated every four weeks until enough plants have been obtained. As a general rule shoots can multiply 3-4 folds every four weeks. The important point to note here is that high rate of contamination during this stage suggests that you are getting air borne contaminants due to poor hygiene.

Root Formation:

Once you have established enough shoots, let them grow to at least 2 cm before beginning the rooting process. Transfer shoots to the rooting medium containing coconut milk and malt. Up to five shoots may be put in each culture vessel. Store containers in their usual place as before. Roots should form within two to four weeks.

Transfer to Potting Mix (Acclimatisation):

The operation at this stage is carried out on the open bench. The rooted cultures can be treated as follows:

  1. Fill the pots with suitable potting mix without any fertiliser and water well. Allow to drain.
  2. Remove the rooted plants from agar medium using a pair of forceps.
  3. Wash off the agar thoroughly from the roots using lukewarm water.
  4. Insert a hole in the middle of the potting mix and gently insert the roots in that hole.
  5. Spray the foliage with a hand spray containing water. These pots can be kept inside a larger plastic containers with a glass cover, out of the direct sunlight. Gradually remove the glass cover but watch for signs of desiccation and if needed use the hand spray to spray water on the foliage.
  6. When the roots are well established and the plants are acclimatised (this should take about 4-6 weeks), they can be given fertiliser and be treated like any other plant. It is advisable to gradually increase the light intensity for the plants too.

Copyright ©1996 Professor Acram Taji

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  • 21 Responses to “ Plant Tissue Culture at Home ”

    1. Devo Mannar says:

      I’m really happy to find your wep page and I appreciate it. Everything that I’ve been searching on the internet about the steps, equipments, chemicals, etc that are maintain on plant tissue culture, I think I get enough info an this instant.
      Thank you.

    2. denewf says:

      Just found your site.
      Well done!!
      Is it possible to propagate ostrich fern from fiddle-head as purchased at the local store.
      Thank you

    3. Vinod says:

      very nice site…
      I would love to do this at home…i need more detailed guidance

    4. shantanu says:

      a step by step description……very helpful….thanks for the alternative to Ms medium and stuff like that….

    5. Rani Alex, says:

      Very helpful, all in one detailed guidance what I was looking for. Thank you very much.

    6. Bruno Donati says:

      So interesting, thank you , I have a plantains plantation here in Costa Rica and I want to start producing high quality seedlings with tissue culture.For sure I need more specific information about how to do things right and would be prepare to economically recompens for assistens.

    7. Doug says:

      Thanks for this, and good chi to you.

    8. Pradeep says:

      how long would it typically take to produce/see new plants in your jar, once you place all the materials ?

    9. Romeo Fenol says:

      is it okay to use your media preparation in this blog for tissue culture of coconuts? please reply via e-mail. thank you.

      P.S. Im from Philippines

    10. bilal sanjrani says:

      Hi dear sir I’m growing banana tisuue culture but I can buy murashige and skoog ms medium chemical I’m a farmer please tell me how can I grow simple and easy tissue culture bananas for my agriculture farm here is chemical very high price in my country Pakistan and tell me how can I make ms medium chemical at my home thanks

    11. P.A.M. Krishanthi says:

      It seems very helpful. Can I get more detail?

    12. Enoch E. Ademuwagun says:

      This is really very helpful to my needs. Can I get more details for the tissue culture of banana and Plantain plants?

    13. Qasim Iqbal says:

      can anyone tell me will it woek with proper sterilizing and good handling?

    14. Mike Daniel says:

      Great! this is what i’ve been looking for. I need more details so as to carry out this process successfully. My email is attached. Thanks

    15. Salim MOHAMMED says:

      very informative thank you

    16. Jakil says:

      What about animal tissure culture ?
      Is it much more complex than this ?

    17. Acram Taji says:

      Thank you for all your comments about Plant Tissue Culture for Home Gardeners. Please note that my comprehensive video on Basic Plant Tissue Culture which was produced in 1997 is now uploaded onto UTube in three parts.

      Part one is at below link:

      https://www.youtube.com/watch?v=Cw4oNRVxwSY

      Part two is on the link below:

      https://www.youtube.com/watch?v=rTEl59gPBjA

      Part three is at:

      https://www.youtube.com/watch?v=gwzqC5HG62A

      One of my PhD graduates kindly put this up for all to access

      Warm regards,
      Acram Taji
      Professor Emeritus

    18. Mohamed asif shaw says:

      I’m studying 11th standard. I’m interested in botany. I wish to do some plant tissue culture at my home. Please guide me to the that technique….

    19. Iam a botanist and i found this article very informative.iam graduate and love to study further to phd level but financially nowadays my family cannot support my studies.iam compelled to go UAE and work as a driver.somebody please enlight my way towards achieving my goal.thank you

    20. Robert Thacker says:

      This sound very good I will try it out and see if I have the skill and Hygene necisary. I have been gardening as a past time for fourty years and want to multiply plants for fun. since 2002 I have been growing veg in rased beds as we have large trees that would other wise take the nutriants. In the next couple of years I intend to try aquaponic growing again for fun so having a way to increase the number of plants fits with this idea. I only have gold fish at this stage.
      Thanks for such an informative site.

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