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Detection of Important Plant Viruses in In Vitro Regenerated Potato Plants by Double Antibody Sandwich Method of Elisa

M. Salim Khan
Tissue Culture Section, BCSIR Laboratories, Dhaka-1205, Bangladesh.

M. I. Hoque
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh.

R. H. Sarker
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh.

H. P. Muehlbach
Department of Genetics, Institute of General Botany and Botanical Garden, University of Hamburg, Ohnhorststr, 18, D-22609 Hamburg, Germany

Abstract/ Executive Summary:

To develop a reliable and sensitive method for virus identification in vitro regenerated plantlets of six potato varieties such as, Cardinal, Diamant, Dhera, Multa, Cilena and Sieglinde were mechanically inoculated with seven potato viruses, namely A, Y, V, M, S, X and PLRV and identified through double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). In vitro regenerated non-inoculated plants were used as negative control. Among the seven viruses used, except PLRV others infected the host tissue mechanically. Although no physical symptoms could be observed in some cases after mechanical infection, the presence of viruses could be detected through DASELISA test. There were no antiserum reactions in the non-infected and in vitro regenerated negative control plants.

Keywords: Potato, Virus detection, DAS-ELISA

Location: All over Bangladesh

Start Date:

End Date:

Program Area: Pest Management

Commodity: Potato

Objectives:

To identify the presence or absence of the virus particles in mechanically infected in vitro regenerated plants with seven viruses, namely PLRV, PVA, PVY, PVV, PVM, PVS and PVX using DAS-ELISA.

Methodology:

Six cultivated potato (Solanum tuberosum L.) varieties of which four, namely Cardinal, Diamant, Dhera and Multa were collected from Bangladesh and two viz. Cilena and Sieglinde were from Germany. Leaf samples were collected from in vitro raised plants of all varieties which were grown in the greenhouse, in non-infected (S1) and infected (S2) chambers. Field samples were collected from the plants showing virus-like symptoms as well as from symptomless ones from Saka- Ragis Pflanzenzucht GbR experimental field, Zuchtstation Windeby 24304, Windeby bei Eckernforde, Germany. The in vitro raised plantlets and microtubers were sown in pots and as well as grown in the greenhouse. Two weeks after sowing, the plantlets were mechanically inoculated at their primary leaves with a crude leaf homogenate of positive control plants infected with PLRV, PVA, PVY, PVV, PVM, PVS and PVX. IgG used in the present experiment was diluted in the carbonate buffer in the proportion of 1 : 1000. Plates were incubated for 3 h at 37⁰C. Following incubation plant extracts were applied on these wells. The plant extracts were prepared with a hand-held rotary grinder in a maceration buffer containing phosphate buffered saline (PBS) with 0.5 ml/l Tween 20 and 2% polyvinyl pyrrolidone. After adding the crude plant extract, the virus was detected by the corresponding antibody conjugated with alkaline phosphate diluted in conjugate buffer (PBS-TPO, pH 7.4) according to the suppliers specifications. Plates were washed with phosphate buffered saline (PBS, pH 7.4) at each stage. Absorbance at 405 nm (measured with a ELISA Reader; DYNARECH MR 50000, U.S.A.) was read 30 min to 3 h after incubation with the substrate p-nitrophenyl phosphate (1 mg/ml, pH 9.8). A sample was considered positive if the absorbance was at least three times greater than that of the healthy control plant (negative control).

Source of Information: Plant Tissue Cult. 13 (1) : 21-29, 2003 (June)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The results of the present study indicate that it is possible to detect PLRV, PVA, PVY, PVV, PVM, PVS and PVX by the DAS-ELISA test from in vitro raised infected and non-infected plants and it may be recommended that the above DAS-ELISA method can be widely used for virus detection in potato and other related plants. As already mentioned the advantage of this assay is that the virus particles are concentrated from extracts by the coating antibody, and the inhibitory components of extracts are removed by rinsing before addition of detecting antibody and enzyme substrate. The limit of detection of most plant viruses in tests on host tissue extracts between 1 and 10 ng/ml when using the chromogenic substrate p-nitrophenyl phosphate (NPP). The product of hydrolysis of NPP by alkaline phosphate (AP), turns yellow in alkaline solution and strongly absorbs light at 405 nm. Substantially lower limits of detection (>100 ∇) can be achieved by using fluorogenic or chemiluminescent substrates.

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