Progressive Education Society's

MODERN COLLEGE OF ARTS, SCIENCE, AND COMMERCE

( Autonomous) Ganeshkhind, Pune - 411 016
DEPARTMENT OF BIOTECHNOLOGY

Subject: 23MBT-306P Laboratory Course in Agricultural Biotechnology

Name: Date
Class: Batch: Remark
Roll No. Exp. No. Signature of
teacher

Title of Exp: Production of virus-free plants (Virus indexing- ELISA and PCR)

Aim: To produce Virus – free plants by Virus Indexing using PCR Technique.

Introduction :
Virus Indexing is a procedure to determine the presence or absence of viruses not readily transmitted
mechanically. Material from a “test” plant is grafted to an “indicator” plant that develops
characteristic symptoms if affected by the viral disease
(https://www.britannica.com/science/indexing).Virus detection is necessary for in vitro production
and propagation of virus‐free plants, because virus diseases cause great losses of crop yield and have
long been a constraint for sustainable developments of agricultural production.(Cui et al. 2015).

There are two types of Viruses :

DNA viruses contain DNA as their genetic material. DNA viruses infecting plants are classified into
two groups according to the Baltimore classification (Muhammad Shafiq et al. 2020) of viruses:
single-stranded (ssDNA) DNA viruses and double-stranded (dsDNA) DNA viruses.

Plant RNA (ribonucleic acid) viruses are obligate intracellular parasites with single-stranded or
double-stranded RNA genome(s) generally encapsulated but rarely enveloped (Carbonell et al. 2016).

Musa species (Banana) is the most important fruit crop in India and plays a major role in the
livelihood of millions of resource-poor small farmers. The use of quality planting materials is very
important for increasing productivity. However, banana viral pathogens, which are economically
important in India, can be inadvertently spread through tissue-culture plants. In order to control the
spread of the viruses, virus-indexing techniques were developed at the National Research Centre for
Banana, Tiruchirappalli for early detection in mother plants and tissue-culture plants used for mass
propagation. Polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), Nonradioactive
probe-based nucleic acid spot hybridization (NASH), and enzyme-linked immunesorbent assay
(ELISA) based techniques were developed and validated for routine virus testing.

Banana bunchy top virus (BBTV):

BBTV has a multicomponent genome comprising six transcriptionally active components
each 1 kb in size. BBTV is systemic within the banana plant, and following aphid inoculation. The
first symptoms comprise a few dark green streaks and dots on the lower part of the lamina and on
the petiole, becoming more general on subsequent leaves. These streaks form hooks as they enter the
midrib, and are best viewed from the underside of the leaf, with transmitted light. Successive leaves
become shorter and narrower, and have a brittle lamina with upturned, ragged margins.

Figure 1 (A) BBTV infected plant showing stunting and bunchy leaves. (B) BSV infected plant
showing yellow streaks.

Banana Steak Virus (BSV):

Banana production is threatened by the Banana streak disease (BSD), and its pathogen
belongs to the genus Badnavirus, the family Caulimoviridae. BSV is a kind of para-retroviruses
(EPRV) that use a virus-encoded reverse transcriptase (RT) to reverse viral RNA (vRNA) into viral
DNA, completing the viral DNA replication process. BSV possesses an open-circular double
stranded DNA genome of 7–8 kb in size and its genome is encapsulated inside non-enveloped
bacilliform particles (30 nm × 150 nm), encoding three open-reading frames (ORFs).
(Jaindra Tripathi et al. 2019). The diversities of complete genome sequences of more than nine BSV
species indicated that the virus is highly variable and polymorphic. Furthermore, it is difficult to
study the invasion mechanism and pathogenesis, owing to the instability of symptoms on the host
and the integration of the BSV genome into the host genome (Selvarajan et al. 2011).
ATL laboratory- Virus Indexing of Tissue Culture Raised Plants:
Vasantdada Sugar Institute(VSI) is working on major objectives viz. DNA marker technology
production of virus free plant and as an Accreditation Test Laboratory for Virus Indexing. VSI is one
of the ATL work under the NCS-TCP guidelines for certification of virus indexing of tissue culture
raised plants. It is involved in virus indexing (VI) and certification of tissue culture raised plants
produced by commercial tissue culture production units (TCPUs) registered with BCIL.

Polymerase Chain Reaction (PCR):

PCR- Polymerase Chain Reaction is an in vitro method of enzymatic synthesis of specific DNA
sequences. This technique was developed by Kary Mullis in 1983. It is a very simple and inexpensive
technique for characterizing analyzing and synthesizing any specific piece of DNA or RNA from
virtually any living organism (plant, animal, virus, or bacteria).
The multiplex polymerase chain reaction (Multiplex PCR) refers to the use of PCR to amplify several
different DNA sequences simultaneously using multiple primers in one tube and one amplification
program for all amplicons (James B. et al. 1995).
Multiplex RT-PCR method is a simple, specific, rapid, reliable, yet sensitive and cost-effective,
diagnostic tool for viruses. The method can be successfully utilized for in detecting all ten viruses
individually as well as in multiple infections from the same plant. A duplex PCR method allows the
simultaneous detection of two viruses in the same sample, which is more cost-effective than assaying
for each virus separately (Carlos Leal et al. 2014).

Materials Required:
1. GeNeiTM PCR kit
2. PCR tubes
3. Sample: Banana plant sample
4. Equipment: Himedia PCR, Gel documentation, Horizontal Electrophoretic Unit, LAF,
Nanodrop spectrophotometer
5. Reagents: D/w, EtBr
6. Other Requirements: Crushed ice, Tips, Gloves, Micropipettes
Procedure:
Setting up PCR
1. Add the reagents to the PCR tube in the following order:

DNA (BBTV+/BSV 2 µl
10X Assay Buffer 2 µl
10 mM dNTP Mix 1.5 µl
Forward primer 1 µl
Reverse Primer 1 µl
Taq DNA Polymerase 0.45 µl
MgCl2 0.5 µl
ddH2O 6.5 µl
Total Reaction Volume 15 µl

2. Carry out the amplification in a thermocycler for 30 cycles using the following reaction
conditions:

Initial Denaturation Annealing Extension Final Extension

Denaturation

940C 940C 570C 720C 720C

5 min 30 sec 45 sec 1 min 10 sec

 For 35 cycles →

Preparation of 1.5% Agarose Gel & Electrophoresis:

1. Prepare 1X TAE by diluting appropriate amount of 50X TAE Buffer (For one
experiment, approximately 200 ml of 1X TAE is required. Dilute 4 ml of 5OX TAE to
200 ml with distilled water).
2. Weigh 3 g of agarose and add to 200 ml of 1X TAE. This gives 1.5% agarose.
3. Boil till agarose dissolves completely and a clear solution is obtained.
4. After cooling to 55°C add 2ul of EtBr.
5. Meanwhile place the comb in electrophoresis set such that it is approximately 2 cm
away from the cathode.
6. Pour the agarose solution in the central part of tank. Do not generate air bubbles. Keep
the gel undisturbed at room temperature for the agarose to solidify.
7. Pour 1X TAE buffer into the gel tank till the buffer level stands at 0.5 to 0.8 cm above
the gel surface.
8. Gently lift the comb, ensuring that wells remain intact.
9. Connect the power cord to the electrophoretic power supply according to the
convention red: anode, black: cathode.
10. Following PCR amplification, add 1 ul of Gel loading buffer to each of the PCR tubes.
11. Tap the mixture thoroughly and wait for a few seconds for the 2 layers to separate.
12. Carefully pipette out 8 µl of reaction mixture and load in the well of the 1.5% agarose
gel.
13. Load 1µl of the DNA ladder (100bp) provided. Note down the order in which the
samples have been loaded.
14. Set the voltage to 50 V and switch on the power supply.
15. Switch off the power when the tracking dye (bromophenol blue) from the well reaches
3/4th of the gel. This takes approximately one hour.
16. Visualize the gel under a UV trans-illuminator.
Fig 2. Schematic diagram of cycle of cleaning and indexing to generate virus-free plants
References

1. Selvarajan, R., Balasubramanian, V., Sheeba, M. M., Raj Mohan, R., & Mustaffa, M. M. (2011).
VIRUS-INDEXING TECHNOLOGY FOR PRODUCTION OF QUALITY BANANA
PLANTING MATERIAL: A BOON TO THE TISSUE-CULTURE INDUSTRY AND BANANA
GROWERS IN INDIA. Acta Horticulturae, (897), 463–469. doi:10.17660/actahortic.2011.897.63

2. Leal, C.A.G., Carvalho, A.F., Leite, R.C. et al. Development of duplex real-time PCR for the
detection of WSSV and PstDV1 in cultivated shrimp. BMC Vet Res 10, 150 (2014).
https://doi.org/10.1186/1746-6148-10-150

3.James B. Mahony, Max A. Chernesky, 10 - Multiplex Polymerase Chain Reaction,Editor(s):

Danny L. Wiedbrauk, Daniel H. Farkas, Molecular Methods for Virus Detection, Academic Press,
1995, Pages 219-236, ISBN 9780127489209,

https://doi.org/10.1016/B978-012748920-9/50011-X.

4. Tripathi, J.N., Ntui, V.O., Ron, M. et al. CRISPR/Cas9 editing of endogenous banana streak
virus in the B genome of Musa spp. overcomes a major challenge in banana breeding. Commun
Biol 2, 46 (2019). https://doi.org/10.1038/s42003-019-0288-7

5. Muhammad Shafiq, Fasiha Qurashi, Sehrish Mushtaq, Mujahid Hussain, Amir Hameed,
Muhammad Saleem Haider, Chapter 13 - DNA plant viruses: biochemistry, replication, and
molecular genetics, Editor(s): L.P. Awasthi, Applied Plant Virology, Academic Press, 2020, Pages
169-182, ISBN 9780128186541,

6. Cui, Z.-H., Bi, W.-L., Hao, X.-Y., Xu, Y., Li, P.-M., Walker, M. A., & Wang, Q.-C. (2016).
Responses of In vitro-Grown Plantlets (Vitis vinifera) to Grapevine leafroll-Associated Virus-3 and
PEG-Induced Drought Stress. Frontiers in Physiology, 7. doi:10.3389/fphys.2016.00203

7.https://doi.org/10.3389%2Ffmicb.2020.609784

8.https://doi.org/10.1111%2Ftpj.14466

9. https://www.britannica.com/science/indexing

10. Maruthi, M.N., Physiological and Molecular Plant

Pathology,https://doi.org/10.1016/j.pmpp.2018.0