Plant viruses are found in nature and propagate inside the cells of living plant. There are many plants that suffer from serious diseases developing a variety of symptoms such as mosaic, chlorosis, necrosis, vein clearing, leaf curling, leaf rolling, etc.
Sometimes the disease severity is so high that viruses migrate to vascular bundles and also get established inside the seeds.
For the first time G. Morel and C. Martin (1952) produced virus-free shoots of Dahlia from virus-infected plants through shoot meristem culture. On 26th November, 2002 a US Patent was granted to H.C. Chaturvedi and co-workers for successfully regenerating and proliferating shoot meristem of Citrus spp.
Using tissue culture method you can produce disease-free plants. Generally, the apical meristem of plants is free from viruses and the other microorganisms. However, if viral particles are present, their number would be quite low.
Methods of Virus Elimination :
There are two methods that are used to produce virus-free plants.
(a) Heat Treatment of Meristem:
Before meristem culture, viruses associated with meristem are eliminated in vitro by heat treatment (thermotherapy) of whole plant. Heat treatment is given through hot water or air at 35-40°C for a varying period (a few minutes to several months).
It should be noted that prolonged heat treatment inactivates the host resistance. Percentage of plant survival is also low. Temperature tolerant viruses may survive inside the plant tissue.
(b) Meristem-tip Culture:
Meristem-tip culture is the most widely applicable approach for virus elimination. In most of the cases explant (shoot tip) of 100-1000 µm has been cultured to raise virus-free plants.
But the explant of small size (1 mm) i.e. meristem tip is preferred for in vitro culture. Meristem tip is excised in aseptic conditions and cultured on nutrient medium. If required thermotherapy is given to the mother plant before excision of meristem tip. The inoculated tubes are incubated properly in light and dark regime.
There are some chemicals which are used to eliminate viruses from plant tissues and protoplasts. These are malachite green, thiouracil, acetyl salicylic acid, cycloheximide, actinomycin- D, etc.
(c) Virus Indexing:
Virus indexing is the testing of plants for the presence or absence of viruses. Each meristem tip or callus or callus-derived plantlets must be tested before using it as mother plant to produce virus-free stock. Following are the methods for virus indexing:
Sap Transmission Test:
It is the most sensitive method which is performed on commercial scale. Cell sap from the test plant (i.e. plant to be tested for presence of virus) is obtained after grinding the leaves. Filtered leaf sap (extract) is put on the indicator plant (i.e. a plant susceptible to a specific virus or group of viruses).
If the indicator plant develops symptoms, the leaf extract contains viral particles. If no symptoms develop, it means that the plant is free from viruses.