Micropropagation: Organogenesis

 Organogenesis

 It is the process in plant tissue culture where adventitious shoots, roots, or other organs are developed. 

Organogenesis has two steps the caulogenesis and rhizogenesis.  Caulogenesis is the formation of the shoot. Rhizogenesis is the formation of the root.

ORGANOGENESIS OVERVIEW

 For plant tissue culture, partially differentiated or poorly differentiated tissues are used. These tissues when grown under suitable conditions develop into the root or shoot.

 Organogenesis involves two cellular processes, dedifferentiation, and Redifferentiation.

 Dedifferentiation is a reversal of differentiation that is partially differentiated or fully differentiated cells become meristematic lose their specialty and now become totipotent. That can divide multiple times and can produce all types of plant tissues. 

Redifferentiation is the differentiation process that is regained by the dedifferentiated cells to undergo organogenesis.

The organogenesis can be direct that is the cultured tissue may directly develop into the shoot bud and which is done subjected to rooting.

 Organogenesis can be indirect where culture tissues undergo the formation of callus and the callus is subcultured to form shooting and his shoot and culture to for root formation.

 Organogenesis can be divided into three steps, callus formation, shoot formation, and root formation. 

 Explants for organogenesis can be any issue from roots, stems, buds, or leaves. The most common tissue used is mesophyll cells of the leaf.

 For this leaves are taken from the plant. These leaves are sterilized using hypochlorous acid or HgCl2, then washed with autoclaved tissue grade distill water. The explant is cut under aseptic conditions into small pieces. These pieces are then inoculated into a culture medium. The cut pieces of leaves start showing the development of callus in 2-3 weeks. This callus can be used for the formation of more calluses or it may be subjected to organogenesis. The callus is cut into pieces and subcultured into a shooting medium. In shooting medium callus develop vascular tissue and result in the formation of buds, which result in shoot formation. 

Rooting requires the transfer of shoots in the rooting medium.

Shooting and rooting medium(1)

 The formation of shoot requires the presence of cytokinin, auxin, and carbohydrates. The hormones act in combination or they may be produced by the tissue itself.

 Where external supply is required NAA and kinetin have shown good results for the development of shoot. Where a high ratio of kinetin and auxin determines the formation of the shoot. A 1.5 mg per liter kinetin in addition to 0.15 or 0.3 mg per liter NAA induces shooting.

 The reduced amount of cytokinin to that of auxin results in the formation of callus.

Shoots are cut and inoculated to form roots in the rooting medium. The rooting medium consists of different types of auxin and cytokinin. A 1.0 mg/ L IBA or 1.0 mg/L NAA induces the production of rooting in incubated shoots. 

When complete organogenesis occurs the plantlet is produced, which has roots and shoots. The plantlets are further inoculated multiple times to achieve a perfect length of plantlet.  These plantlets are then allowed to harden. For hardening, they are cultured in soil under aseptic conditions. If the plant survives in the soil they are then cultured in poly farms for further hardening, if they survive there are also, they are cultured in the field.

 

Factors affecting organogenesis

 Organogenesis is affected by internal phytohormones present in cultured tissue.

 If the plant has the internal level of auxin and cytokinin and is appropriate for the formation of bud it will form bud without an external supply of these hormones.

 Again the level of cytokinin to auxin determines the formation of organ whether the tissue will develop into to shoot or root.

The presence of vascular tissue in cultured explant also favors the formation of the shoot in cultured tissue.

 The presence of 2,4-D along with a low level of cytokinin induces callus formation in the cultured tissue.

 High intensity of light increases the formation of callus.

 Regular light and dark periods induce the formation of the shoot in culture tissue.

 Ethylene blocks the formation of shooting in cultured tissue.

Kinetin along with NAA found to be more appropriate for induction of shooting in the cultured plants.

Material and methods

Organogenesis involves following steps,

Preparation of instruments

Preparation of medium

Preparation of explants

Inoculation of explants

Inoculation for shooting

Inoculation for rooting

Hardening

Preparation of instruments

Instruments required for plant tissue culture are scissors, blades, blade handles. These are autoclaved.

Glasswares i.e. flasks, Petri plates, tissue papers, beakers, pipette, and pipette tips, test tubes are washed and packed in aluminum foils and autoclaved, along with tissue grade distilled water.

Preparation of medium

Prepare stocks of MS medium.

Use powdered MS medium.

Weigh 12 g of MS medium.

Dissolve it in 800 ml of tissue-grade water.

Add sucrose to it.

Dissolve gelling agent (agar) in 200 ml of water by heating.

Mix the two solutions and maintain the pH at 5.4 to 5.8.

Autoclave the medium in a flask plugged with a cotton plug.

Pour the medium in culture vessels under aseptic conditions.

Allow it to cool.

Preparation of explants

Take the explants. It must be taken from healthy, disease-free, and actively growing plants.

EXPLANTS

Wash with water and mild detergent.

 Sterilize it with chemicals. Antibiotics are also used for sterilization.

Wash with sterilized water.

Cut into small pieces.

Inoculation of explants

CUTTING OF EXPLANT

Prepared plant tissue is inoculated under aseptic conditions in a culture medium. A callus will develop at the cut ends of the explants in 2-3 weeks.

INOCULATION OF EXPLANT

Inoculation for shooting

SHOOTING

The callus is cut into pieces and inoculated in a shooting medium. Buds will appear in a week. The shoot will develop within two weeks. It is subcultured to separate it from the callus and obtain an appropriate height of shoot.

Inoculation for rooting

ROOTING

Shoots are inoculated to rooting medium for rooting.

In this way, a plantlet is obtained.

This plantlet is culture to obtain a full-length plant. These are then subjected to hardening.

 

Hardening

PLANTING IN POTS

 Plantlets are cultured in pots and subjected to hardening.

Where these plantlets are cultured in greenhouses and hardened for temperature, pH, light intensity, etc.

Hardened plants are then cultured into fields.

PRECAUTIONS

Aseptic conditions should be maintained during cutting, inoculation, media preparation, and pouring.

Laminar airflow should be used as the working platform.

It should be sterilized using UV radiation before working.

UV should be switched off before you start working in LAF and the fan should be switched on while working.

Ethanol should be used to sterilize hands and instruments while culturing.

Surgical blades are used for cutting.

Metal instruments should be sterilized by flaming.

Keep a record of your culture experiments.

After inoculation, close the culture vessels tightly and place them in the culture room.