Virus-free Seedling Production of Bhut Jolokia Chilli Using Tissue Culture Techniques

Abstract

Mosaic, dwarfing, ring spot, leaf curling and necrosis were indicated as the symptoms of viral diseases found in Bhut jolokia chilli grown at Pangda Station, Royal Project Foundation. Various techniques were applied to diagnose the majority mosaic symptom. The positive results of virus transmission by shoot grafting of infected plants on healthy stock plants were observed. For microscopic symptom observation; the micrographs of crystalline inclusion, cylindrical inclusion and amorphous inclusion in the infected plant were detected after the epidermis and vascular leaf cells were stained with Azure II, orange green, toluidine blue and acridine orange. A total of 60 lateral and shoot buds of the virus infected Bhut jolokia chilli plant were surface sterilized and cultured in vitro on MS medium. After 3-6 months, the mosaic symptom plantlets even in the second generative propagations were randomly diagnosed for a particular virus infection. However, thermotherapy and meristem tip culture for virus eradication procedures were conducted under two treatments of 35 ±1 ^oC and 38 ±1 ^oC using 40 infected plantlets per treatment. Total of 13 and 8 survived plantlets were obtained from 35 ±1 ^oC continuously treated conditions for 4 and 8 weeks respectively. The meristems of these survived plantlets were excised in 0.5 mm in size, then cultured in MS medium supplemented with 0.04 ppm of BAP and 0.1 ppm of GA₃. Three pieces of the meristem were successfully regenerated to complete plantlets while 13 pieces were generated to be calli and 5 pieces were necrosis deaths. The survived plantlets were grown and subcultured on MS medium. Viral symptoms on the plantlets grown in vitro were not found during the long term period of cultures. Diseased free plantlets were propagated on MS medium containing 10 ppm of BAP and 1 ppm of kinetin which produced 4.5 shoots per axillary bud in one month. The separated shoots were rooted successfully on MS medium containing 2 ppm of IAA. Acclimatization was carried out by transferring the bottle of in vitro plantlets to normal condition in greenhouse for 7 and 14 days with survival percentages at 85 and 90 of transferred plantlets in sterilized compost were observed.