Pathogen/Virus Free Plants

Most of the crop plants propagated by vegetative means, are systemically infected with one or more pathogens. Strawberry plants are known to be attacked by 62 viruses and mycoplasmas Pathogen attack does not always lead to death of the plant. Many viruses may not even show visible symptoms. But, the presence of viruses in plants reduces the yield and/or quality of crops. Yield increases up to 300% (averaging 30%) have been reported by replacement of virus-infected stock with specific pathogen-free plants

The reasons proposed for the escape of the meristem from virus invasion are (a) viruses readily move in a plant body through the vascular system which is absent in the meristem. The cell-to-cell movement of the virus through plasmodesmata is too slow to keep pace with the actively growing tip (b) high metabolic activity in the actively dividing meristem cells does not allow virus replication (c) the 'virus inactivating systems' in the plant body, if any, has higher activity in the meristem than in any other region; thus, meristem is protected (d) a high endogenous auxin level in shoot apices may inhibit virus multiplication.

Virus free Specific virus free or specific pathogen free

Virus eradication

Thermotherapy Chemotherapy Meristem/shoot tip culture

Thermotherapy

Principle - at temperatures higher than normal many viruses in plant tissues are partially or completely inactivated with little or no injury to the host tissue Explant/whole plant exposed to higher temp, lethal only to viruses. 38-52 C for few min to several weeks. Virus encoded movement proteins are temp sensitive. High temp disrupt proteins Coat protein also disrupts as it is also temp sensitive. Interferes in virus replication.

The area B-C is critical in heat treatment; the greater the spread between the thermal death point of the host (C) and the parasite (B), the greater are the chances for successful thermotherapy

Thermotherapy: methods

Hot Water Hot air In vitro heat treatment Mother plants may be grown at elevated temperatures. Adequate supply of humidity and light should be maintained during heat treatment. The plants must have ample carbohydrate reserves

Thermotherapy: Limitations

Not all viruses are sensitive to the treatment. Prolonged heat treatment inactivates plant resistance system Only isometric and thread like virus are eliminated through thermotherapy Survival rate low.

Chemotherapy

Virus elimination by chemicals. Actinomycin D, Ribavirin, 2-thiouracil, Cyclohexane, Malachite green etc Added in medium at particular conc. Interferes in virus replication Also cytotoxic to plants, so used in slightly less concentrations

Both Thermotherapy and chemotherapy, although helps in eradication of viruses but has certain limitation.

Meristem Tip Culture

Holmes (1948), described gradient of virus distribution in shoot tips. Produced virus free plants of Dahlia through shoot tip culture. Mainly used for virus elimination, meristem-tip culture enables plants to be freed from other pathogens (viroids, mycoplasmas, bacteria and fungi)

Meristem Tip Culture

Meristem: Localized group of actively growing and dividing cells from which permanent tissues are derived. Apical meristem: Group of cells situated at the extreme tip of root or shoot in the shape of dome. 100 m in diameter and 250 m in length, remains in active state and always young. Shoot apex: length upto 500 m, have leaf primordia Meristem tip prefered over shoot tips.

Meristem Tip Culture

Meristem cells are highly conservative and are genetically stable and hence plants produced are genetically stable. Rate of cell division is very high Absence of plasmodesmata and vascular bundles.

Meristem Tip Excision

Stem apices or tuber sprouts, protected with overleaf primordia. Excision of Meristem: under aseptic conditions, stereomicroscope or light source required, excise active meristem, prevent it from dessication (cover in wet paper), sterilize by 70% ethanol for few seconds before culture. PGRs helps in virus eradication eg. Cytokinins interfere virus replication

Callus cultures for virus elimination : Shoot tip grafting or micrografting:

extremely beneficial for woody plants.

Very small tip consisting of meristem with 2-3 leaf primordia to the in vitro grown virus free root stock.

Citrus, apple, prunes etc

Virus Indexing or Virus Detection

Sap

Transmission or Bioessay or Infectivity test: Serological Detection (ELISA) Nucleic acids Based: hybridization or PCR Electron Microscopic studies

Applications