Eradication of Cymbidium mosaic virus from protocorm–like bodies of Dendrobium Shavin White ‘White 5N’ using cryo-treatment

Abstract

The main problem in vitro propagation of the Dendrobium hybrid was that almost mother plants were infected with the Cymbidium mosaic virus (CymMV). The critical problem in producing virus–free plantlets using meristems culture was the slow growth and high mortality rates. Using protocorm–like bodies (PLBs) derived from virus–infected young shoots to produce virus–free orchid plants might have good potential. This study aimed to eliminate infected–CymMV from PLBs of Dendrobium Shavin White ‘White 5N’using the encapsulation–dehydration method with cryo-treatment. The 3 mm PLBs from CymMV–infected young shoots were pre–cultured in liquid Vacin and Went (VW) medium supplemented with 15% coconut water and 0.15, 0.175, and 0.2 M sucrose, each concentration for 1 day sequentially. After 3 days, the mixture of sodium alginate solution with PLBs is dropped with a sterile pipette into CaCl₂ solution and maintained for 20 min to form beads and dehydrated in a laminar airflow for 5 h. The dehydrated beads were transferred to cryotube and immersed directly in liquid nitrogen (LN) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 or 30 sec and 1, 3, 5, 10, 30, 60 or 90 min. The experiment was a completely randomized design. After LN immersion, cryotubes were immersed in a 40°C for 5 min, and the beads were transferred to culture in a modified semi–solid VW medium supplemented with 15% coconut water and 2% sucrose for 6 weeks. The cryo–treatment resulted in a significant decrease in the PLBs survival rate from 1–4 weeks (P≤0.01). All PLBs were dead after being immersed in LN for over 10 sec, and the survival rate after 1–5 sec immersion was 55–70%. The regrowth rate comparison found that the highest obtained from 1–7 sec immersion were 62.50–88.89% (P<0.01). The surviving PLBs can successfully grow into plantlets. However, CymMV was still found