Production of Virus-free Shallot Using Tissue Culture Techniques

Abstract

Shallot cv. Ban-Hong, bulb seeds were collected from planting area of Ban Hong district, Lamphun province. The samples were planted in sterile soil for 20 days under greenhouse condition before viral symptoms on the leaves were evaluated. Five levels of disease severities were recorded in number 0-4 (no symptom - severe symptoms). Results of the yellow stripe symptoms were detected on the leaves of all the samples, no healthy normal sample was observed. Disease severities, level 2 was the most detected at 37% followed by the level 1, 3 and 4 at 28.5, 18 and 16.5%, respectively. The virus types were diagnosed with various techniques. The mechanical transmission using infectious leaf sap was first detected and the results revealed local lesions on leaves of indexing hosts, Chenopodium amaranticolor, C. quinoa, Celosia argentea and Cassia occidentalis after 14-20 days of inoculation. Secondly, the commercial POCy KIT was used for serological diagnosis of Potyvirus, Odontoglossum ringspot virus (ORSV) and Cymbidium mosaic virus (CyMV). Positive results for Potyvirus and CyMV in all tested samples were observed and then the positive result was confirmed again by ELISA technique with Potyvirus antiserum from Agdia Elkhart, Indiana, USA. In addition, virus particle was examined under transmission electron microscopy (TEM), flexuous particles of about 10 nm in width and 600-760 nm in length of the particular group of Potyvirus were observed. Thus, meristem tip cultured on MS medium was used to produce the virus-free plantlets. By excised the meristem tip from the bulbs and then ELISA technique was used to evaluate the virus-free plantlets. Results showed 88% of the 0.5 mm meristem tip excisions gave virus-free plantlets with the survival rate at 78.62%. Even, all of the 0.3 mm meristem tip excisions revealed virus-free plant but only 16.78% of that was survived. In order to mass-produce the virus-free plantlets, multiple shoot inductions were tested using various concentrations of plant growth regulators supplemented in MS medium. Results revealed after cultured for 2 months, the 4.2 shoots per explant were successfully developed on the medium supplemented with 2ip at the concentration of 1 ppm and the 2.3 shoots per explant were induced in 0.5 ppm of the same plant growth regulators. On the other hand, shoot multiplication trials on the media mixed with NAA at all concentration showed non significant number of shoot per explant compared with control treatment.