GENERATION OF VIRUS FREE PLANT

 

The vegetatively propagated plants are systematically infected by pathogens. This reduces the yield of plants. The yield increases up to 300% if Virus-free stocks are developed ( Murashige 1980). Viruses are not transported through the seeds, but genetic variations are carried when plants are propagated through seeds. To produce plant clones vegetative propagation is used.  The virus-free plants are produced through plant tissue culture by generating a plant through the vegetative part of the plant. It has been observed that apical meristems are usually free from pathogens ( Quack 1977, Wang and Hu 1980).

Pathogen free plant culture or generation of virus-free plant: Meristem culture

The older tissues are always infected by viruses and other pathogens. The presence of viruses/pathogens increases as we go down or away from the Meristem. It has been proposed that viruses/pathogens are transported through the vascular system so they are also called systemic pathogens. As the vascular system is not developed in the Meristem so there is a reduced chance of the presence of virus in meristematic tissue. Cell to cell transport of viruses occurs through plasmodesmata which are very slow. The presence of a high concentration of auxin inactivates the virus in meristematic tissue. So, meristem culture is used to produce pathogen-free/virus-free plants.

The treatment of viruses

 The light and high-temperature treatment of tissues reduce or inactivate viruses present in the plant tissue (Baker 1962). High-temperature treatment is given to plants using hot water or hot air. Heat treatment has been found effective against isometric and thread-like viruses and diseases that are caused by mycoplasmas.

PRODUCTION OF VIRUS-FREE/ PATHOGEN FREE PLANTS

EXPLANT

Explant of virus-free plant generation is apical Meristems, the shoot tip Meristems that measures 100 μm in diameter to 250 μm in length. The explant is taken with 2-3 primordial leaves, that constitute shoot Apex.

STERILIZATION OF EXPLANT

The explant is surface sterilized by dipping into 70% ethanol and sterilizing with 0.1% sodium hypochlorite for 10 minutes. Fleming after dipping into 95% ethanol has been used for garlic. 

CUTTING

The explant is cut under a microscope using forceps and a needle. The leaf primordia are removed, topical Meristem is cut using a separate sterile needle.  cultured into the medium under aseptic conditions. 

Shoot tip meristem with leaf primordia

INOCULATION

After cutting, the explant is inoculated in an MS medium.

It is cultured for shooting and rooting to obtain plantlets.

The plantlets are mapped for the presence of pathogens.

If the plantlets are found to be pathogen-free these are then used for production stocks of pathogen-free plants.

Clockwise from the above; Explant, Sterilization of explant, Cutting, Inoculation, shooting, rooting, hardening

FACTORS AFFECTING THE GENERATION OF VIRUS-FREE PLANT

 Factors affecting Virus-free plant culture medium Morey, White, and MS media are widely used for tissue culture. Potassium and nitrogen in tissue culture are balanced. MS medium has been found good for Meristem culture as healthy shoots are produced in this medium (Kartha, 1975; Quak, 1977; Wang and Hu, 1980). 

The sugar concentration for meristem culture is 2-4 percent. 

The length of 240 μm the explant develops into a callus.

 BAP and NAA have been shown to be essential to raise full plants from excised Meristem.

The size of the explant 

A complete plantlet is produced from an explant of 200 μm, but an explant smaller than this size will result in the formation of callus. If two or three leaf primordia remain intact, this ensures natural auxin supply to the growing explants ( Smith and Murashige 1970).

LIGHT AND TEMPERATURE

 Light incubation results in the production of plants ( Huth and Bode). Heat and temperature treatment Meristem culture improves the efficiency and effectivity of virus-free plant tissue culture. 

The diurnal low temperature and high-temperature cycle improve the generation of pathogen-free plants in the plant that is sensitive to high temperatures where 40-degree Celsius is given for 16 hours and 22-degree Celsius is given for 8 hours. This eliminated CMV from Nicotiana rustica( Walkey, 1978).

 Maintenance of stock virus-free plant

Stock is maintained in sterile soil in greenhouse or insect Proof cages. Multiple subcultures are used to maintain the stock of virus-free plants.