Plant  Tissue Culture & Orchid seed sowing for hobbyists

by Mano Fonooni

If you have very limited and valuable orchid seeds ,the media and chemicals in this manual are not suitable  . In this case you can substituted  media with orchid medium.

Micropropagtion 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.

All research laboratories  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.
Or you can buy glove bags.
               
You can buy Glove bags from  sigma aldrich     http://www.sigmaaldrich.com/labware/products/aldrich-atmosbag.html
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 for use in surface sterilisation of plant material.
9 - Any skin disinfectant  (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.
If you have very limited and valuable orchid seeds ,the media and chemicals in this manual are not suitable  . In this case you can substituted  media and chemicals with orchid maintenance medium.
 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).
You can buy orchid  media from  ORCHIDSOURCE   http://www.orchidsource.com/GB_catalog.html
 OR from  PhytoTechnology Laboratories   http://www.phytotechlab.com/

Sterilising Instruments and Other Items

Forceps and scalpels can be sterilised by being wrapped in Al. foil and cooked in the pressure cooker for 15 minutes. These items can also be sterilised by being washed in chlorine solution .
PRESSURE COOKER, SANICLAVE 100 STERILIZER  Product ID: S736
Storage Temperature: RT

PhytoTechnology Laboratories

Description:
► Complete Cycle (Warm Up & Sterilization) in 45 min or Less
► Internal Temp: 122° C
► Pressure: 16 psi
► Self-Contained Electric (120 VAC) Heating Element
► Fully Programmable
► Digital Display
► Removable Stainless Steel Chamber and Cover
► Fusible Plug Pressure Release
► Over-Temperature Thermal Cutout 
 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 šC 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 clorox (1/4 cup bleach + 3/4 cup of water + 1 drop of detergent- detergent acts as surfactant). Put plant or orchid green pod ( attempting to sow green pods the first time around, as success rates are highest when you start with a sterile capsule.later discuss the cleaning of contaminated seed. ). 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.
DRY SEED SOWING TECHNIQUES 

Once a seed pod has opened and spilled its seed, or split open even slightly, you must assume that the seed is now contaminated with bacteria or spores of fungus. The biggest problem is fungus spores. Spores are coated with an impervious coat that protects them from chemicals and heat to a great extent. In order to sterilize the seeds of orchids, you must expose the seeds to a sterilant capable of killing the live bacteria and bloomed fungus, but not kill off the seeds at the same time! There is a number of chemicals used but it is possible  to get  excellent success with common household bleach. The trick is to expose the seed to the solution long enough to kill the bacteria, but not the seeds. About  5 percent V/V of household chlorox made with sterilized distilled water, when exposed to the contaminated seeds for a total time of 10 minutes and followed with a double rinse of sterile distilled water works in 70 or 80 percent of all contaminated seed sowing cases.


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:

One of these three things will happen during this time: some of the shoots (seeds) will be killed by the chlorine solution and or the toxin produced by plants themselves; some will be contaminated by fungi and bacteria; or seeds get green and larger , 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: