11 Feb 2021

In Vitro Virus Elimination Techniques

Anjali Singh, MS

As a content and community manager, I leverage my expertise in plant biotechnology, passion for tissue culture, and writing skills to create compelling articles, simplifying intricate scientific concepts, and address your inquiries. As a dedicated science communicator, I strive to spark curiosity and foster a love for science in my audience.

Table of Contents

The Basics of a Virus

The tissue culture of plants is a sensitive and technical process. Moreover, maintaining an aseptic condition is a primary requirement at all steps of the procedure. Generally, we talk about different contaminants, like bacteria, fungi, and mycoplasma. But what about viruses?! A virus is submicroscopic organism replicates very fast inside living organisms. Different viruses infect different hosts and use their machinery for their replication process.

In the previous article titled “Methods to detect viruses in explants,” we talked about all the different processes required to identify if there are any viral infections in the mother plants whose explants you are going to use for your consecutive tissue culture processes. The processes include biological methods (like woody indexing), serological methods (like enzymes linked immunosorbent assay [ELISA]), and molecular methods (like polymerase chain reaction[PCR]).

What happens after you've identified the specific viral organism in your mother tissue? What can you do? After identification, the next step is the elimination of the viruses.

How to Eliminate a Virus

You can eliminate them using different techniques like thermotherapy, meristem tissue culture, in vitro micrografting, in vitro chemotherapy, and cryotherapy of shoot tips, followed by shoot-tip tissue culture or in vitro micrografting. These techniques not only eliminate viruses but they are efficient in eliminating viroids and phytoplasmas as well.

Now the next question that follows is, what are these techniques and how can you use them to eliminate viruses from the explants?

Hold on, hold on! This article is going to answer all your questions. It will discuss in brief some of the techniques mentioned above. Let’s get started!

1. Thermotherapy

It’s also called heat therapy and the oldest of all the methods we talked about. It has been in use since the end of the nineteenth century. Thermotherapy utilizes heat to eradicate viruses from our plants. Often, 35-42 ℃ heat is given to the target mother plant for 4-6 weeks. The duration of treatment and the temperature set up depends on the type of virus, the infected host, and both in combination. This technique has been used to eliminate viruses from plants like potatoes, grapevines, stone fruit, citrus, pome fruits, and strawberries.

The key factors that affect the thermotherapy include temperature and duration, size of shoot tips, source of explants and position of shoot tips, type of virus and infection status, and genotype-specific responses

2. Low-temperature Therapy

It has been observed that the treatment of infected plants at higher temperatures can only eliminate viruses but not viroids. In this therapy, plants are grown at 8 ℃ (compared to thermotherapy in which plants are treated at 37 ℃). This treatment is effective against Potato spindle tuber viroid (PSTVd), Apple scar skin viroid (ASSVd), and Hop latent viroid (HSVd). For infected Chrysanthemum plants {with diseases like Chrysanthemum stunt (ChSVd), Chrysanthemum chlorotic mottle (ChCMVd), and Cucumber pale fruit viroids (CPFVd)} 5 ℃ is the suitable temperature to eliminate viroids.

3. Meristem culture

The shoot apical meristem of plants generally free from pathogens and serve as a perfect explant to tissue culture the choice of plants. The shoot apical meristem is composed of leaf primordia and the apical dome (place of stem elongation). The meristem culture required excision of suitable explanation from the infected parent plant and culture in suitable culture conditions. It’s one of the most widely used technique to eliminate viruses from plants.

This technique has been successfully used to eliminate viral diseases like Onion yellow dwarf (OYDV), Shallot latent viruses (SLV), and Sugarcane yellow leaf virus (SYLV), from the infected plants. You can read more about meristem culture here.

4. In vitro Micrografting

In this technique, meristem tips are grafted on a virus-free rootstock. The grafting technique is joining the two plants as one. A wound is created in one plant and the other plant part is inserted into that. This technique has been used to remove viruses from different plants and trees including peach, almond, citrus, stone fruits, apple, pear, cashew, and avocado.

5. Chemotherapy

Chemotherapy, as the name suggests, involves the use of certain chemicals in the tissue culture media that prevent virus replication or its movement from infected to healthy parts of the plants. Some examples of chemicals are acycloguanosine, azidothymidine, acyclovir, ribavirin, or 2-thiouracil.

This technique is generally combined with other techniques for the most effective results. For example, Potato virus S (PVS) has been eliminated from potatoes by chemotherapy of axillary bud tips.

6. Cryotherapy

This technique involves the cryopreservation (preserving and storing material in liquid nitrogen) of shoot tips and regenerating it later for multiplication. Meristematic tissues are free from viruses and have lower water content than other differentiated tissues. Thus, the ice crystals formed during cryopreservation doesn’t damage the meristem cells. This technique has been successfully used to eliminate viruses like Banana streak virus (BSV), Potato leafroll virus (PLRV), and Grapevine virus A (GVA).

The above-mentioned techniques are combined with one or more techniques for more effective elimination of viruses from the explants.

References

1. Hu, G., Dong, Y., Zhang, Z. et al. Virus elimination from in vitro apple by thermotherapy combined with chemotherapy. Plant Cell Tiss Organ Cult 121, 435–443 (2015). https://doi.org/10.1007/s11240-015-0714-6
2. Varveri, C., Maliogka, V. I., & Kapari-Isaia, T. (2015). Principles for Supplying Virus-Tested Material. Control of Plant Virus Diseases - Vegetatively-Propagated Crops, 1–32. doi:10.1016/bs.aivir.2014.10.004
3. https://www.plantcelltechnology.com/blog/methods-t...
4. https://plantmethods.biomedcentral.com/articles/10...