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5.7. Virus elimination through callus culture
It is a general observation that not all the cells in a calli uniformly carry the pathogen when raised from infected tissues. The two possible reasons for the escape of some cells of a systematically infected callus from virus infection are: (a) virus replication is unable to keep pace with cell proliferation, and (b) some cells acquire resistance to virus infection through mutagenesis. Therefore, it is possible to raise virus-free plants from infected shoot-tip calli. However, genetic instability of cultured cells and lack of plant regeneration in callus cultures of some plants poses the limitations of using calli for virus elimination.
6. Virus indexing
Even after subjecting the meristem-tips to various treatments favoring virus eradication, only a proportion of the cultures yield virus free plants. Therefore, it is required to test all plants, regenerated through meristem-tip or callus cultures, for specific viruses before being used as mother plant to produce virus-free stock. The individual plants consistently showing negative results for virus titre can be marked as ‘virus tested' for specific virus/es and can be released for commercial purposes. The following tests can be performed for virus testing:
i. The simplest test for the presence or absence of viruses in plant tissues is to examine the leaves and stem for the visible symptoms characteristic of the virus.
ii. Another test is the sap transmission test or ‘bioassay test' or ‘infectivity test'. It is a very sensitive test and can be performed at a commercial scale. To perform this, ground the test leaves in equal volume (w/v) of 0.5M phosphate buffer using a mortar and pestle. Leaves of the indicator plant (a plant very susceptible to specific viruses), dusted with 600-grade carborundum, are swabbed with the leaf sap from the test plant. After 5 min the incubated leaves are gently washed with water to remove the residual inoculum. The inoculated indicator plants are maintained in a glasshouse, separate from other plants. It may take several days to several weeks, depending on the nature of virus and the virus titre, for the symptoms to appear on the indicator plants. It is used to detect some viruses and viroids but is a slow process requiring several days to months.
iii. The third method, enzyme-linked immunosorbant assay (ELISA), is more rapid serological test which allows quick detection of important viruses. It relies on the use of antibodies prepared against the viral coat protein, requires only a small amount of antiserum and can be performed with simple equipment. However, it is not applicable to viroids and viruses which have lost their coat proteins