Virus Free Plants - Meristem Tip Culture

 

The plants infected with bacteria and fungi can be treated by bactericidal and fungicidal compounds, there is no commercially available treatment to cure virus-infected plants. A large numbers of viruses are not transmitted through seeds. Therefore, it would be possible to obtain virus free plants from infected individuals by using seeds as propagules. However, genetic variation often occurs from the sexually reproduced plants when propagated by seeds.

Generally, clonal multiplication of cultivars can be achieved by vegetative propagation. However, where the entire population of the clone is infected the only way to obtain pathogen-free stock is to eradicate the pathogen from vegetative parts of the plants and regenerate full plants from such tissues. Once pathogen free plants are obtained they can be multiplied indefinitely under conditions which would protect them from chance reinfection.

It is well known that the distribution of viruses in plants is uneven. In infected plants the apical meristems are generally either free or carry a very low concentration of the viruses. The reasons for low concentration of virus in apical meristerms are:

  • (a)  Viruses readily move in a plant body through the vascular system which is absent in the meristem.

(b) Meristems are always in a state of continuous division because of which virus cannot replicate in such a short period.

(c)  High metabolic activity in the actively dividing meristem cells does not allow virus replication.

(d)  A high endogenous auxin level in shoot apices may inhibit virus multiplication.

Methods of Virus Elimination in Plants:

Often, apical meristems are not always free of virus and it can't be considered as a universal occurrence. There are certain viruses like, Tobacco Mosaic Virus (TMV), Potato Virus X (PVX) and Cucumber Mosaic Virus (CMV), which invade the meristematic region of the growing tips and interrupts the growth of the meristematic tissue.

Different methods are used for elimination of viruses:

Thermotherapy:

 The meristematic dome of the shoot tips is not always free of viruses. Some of the viruses that invade the meristematic region are carnation mottle virus, TMV, potato virus X, cucumber mosaic virus and odontoglossum ringspot virus (ORSV). In such cases, meristem-tip culture alone is not effective in virus elimination. A combination of meristem-tip culture and thermotherapy has been helpful in raising virus-free plants in many such cases.

Shoot tips or nodal explants from infected plants are cultured in vitro after proper sterilization and subjected to heat therapy at 30–400 C in a thermal chamber for varying periods. The new shoots that appear are then used for isolating the meristem-tip and cultured on appropriate media for growth and development.

 Alternatively, meristem-tips are excised from mother plants and subjected to thermotherapy. Apple cultivar ‘Idared’ could be made free of ACLSV and ASPV, following the in vitro thermotherapy technique. This technique is regularly followed for virus elimination in strawberry plants.

Mother plants are exposed to heat treatment before excising the meristem-tips. Thermotherapy of the mother plants prior to meristem-tip culture has an added advantage. It allows initiating cultures with comparatively larger meristem-tip explants, which greatly increases the proportion of explants surviving and developing into virus free plants.

Potato Virus S (PVS) and Potato Virus X (PVX), which are not readily eliminated by meristem-tip culture or thermotherapy alone, could be eradicated by taking meristem-tips from heat-treated mother plants.

 Chemotherapy

Chemical control of viral diseases under field conditions has not been successful so far. However, application of a range of chemicals, such as antibiotics, growth regulators, amino acids, purine and pyrimidine analogues, has met with some success in inactivation of viruses and inhibition of virus multiplication in tissue cultures.

 So far, ribavirin (1-b-D-ribofuranosyl-1,2,4- triazole-3-carboxamide; trade name ‘Virazole’), a base analogue, has proved to be the most effective compound against plant viruses. This broad-range antiviral compound has been shown to be effective in eradicating a large number of plant viruses, such as PVS, PVX and PVY from potato, Chlorotic Leaf Spot Virus (CLSV;) and ASGV  from Apple, PNRSV from plum, ORSV and Cymbidium Mosaic Virus (CyMV) from Cymbidium and Sugarcane Mosaic Virus (SCMV) from sugarcane.

 In sugarcane, 50 mg l-1 ribavirin combined with meristem culture raised the frequency of virusfree plants from 95 to 62 % with meristem alone (Balamuralikrishnan et al. 2002). Apple Stem Grooving Virus (ASGV), which could not be removed by thermotherapy, was successfully eliminated by culturing shoots of Malus domestica on a medium containing 10 lM each of two viricidal compounds, ribavirin (a base analogue) and quercetin (a flavonoid) for 9–12 weeks

Cryotherapy

 Cryotherapy is a novel application of plant cryopreservation techniques to eradicate pathogens, such as viruses, phytoplasmas and bacteria, at a high frequency by a brief exposure of shoot tips to liquid nitrogen using cryopreservation protocols.

 Since the first demonstration of Plum Mosaic Virus (PMV) elimination from an interspecific Prunus rootstock by cryotherapy of shoot tips). This technique has been successfully applied to eradicate Cucumber Mosaic Virus (CMV) and Banana Streak Virus (BSV) from Musa, Grapevine Virus A (GVA) from grapes, PLRV and PVY from potato and Sweet Potato Chlorotic Stunt Virus (SPCSV) and Sweet Potato Feathery Mottle Virus (SPFMV) from sweet potato.

 In cryotherapy, shoot tips (up to 2 mm long) are precultured, directly or after encapsulation in Ca-alginate, in increasing concentration of sucrose (0.25, 0.5 and 0.75 M for 1 day each) to impart desiccation tolerance, vitrified for dehydration and plunged into liquid nitrogen, at -196 C, for about 1 h. The shoot tips are retrieved from the super low temperature by rapid thawing in hot water (40 C) for about 3 min and cultured on a suitable medium for shoot growth and rooting. The plants thus obtained are indexed for viruses.

The explanation offered for the elimination of viruses in the cultures of larger shoot tips following cryotherapy is that exposure to -196 C kills the older, highly vacuolated cells, which carry the infection, and the smaller, highly compact cells with high nucleus to cytoplasm ratio in the apical meristem, which are generally free of viruses, are able to grow and regenerate healthy plants.

 Electrotherapy

Electrotherapy has been used for elimination of PVX from potato, CMV from banana and Tomato Yellow Leaf Curl virus (TYLCV) from tomato.

The technique of electrotherapy involves exposure of shoots to electrical impulses (5–15 mA) for 5–10 min. The shoots are then surface sterilized and inoculated in a suitable medium for regeneration. The regenerated plants are indexed for viruses.

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