Micropropagation and establishment of mite borne virus-free garlic (Allium sativum)

An increasing yellowing in field cultivations of garlic produced by meristem-tip culture two decades ago, maintained under screenhouse and tested in ELISA by polyclonal antibodies, was observed in Italy. Electron microscopy observations revealed the presence of elongated virus particles in both, field and greenhouse grown, garlic plants. This indicates that the material was either reinfected over the time or it was not truly virus-free. RT-PCR tests were developed for onion yellow dwarf, garlic carla- and mite-borne viruses; only primers to mite-borne virus were able to amplify the 280 bp expected fragment from both symptomatic field-collected and greenhouse grown garlic material. New meristem-tip cultures were therefore initiated in order to produce virus-free propagation material; RT-PCR test was employed to evaluate mite-borne virus presence during the micropropagation process. After at least one year in micropropagation, 43 mericlones were tested and 10 (27%) resulted free from mite-borne virus. Among the 79 mericlones in micropropagation for 4-6 months 17 (22%) were free from this virus. The mite borne virus-free mericlones were employed for proliferation tests on MS culture media containing NAA 0.1 mg/l, GA3 0.5 mg/l and TDZ 0.5 mg/l. The ratio obtained ranged from 1:2 to 1:10 according to the mericlone. Proliferated shoots were induced to produce small bulbs and reestablished in insect-proof greenhouse under controlled environmental conditions. A few of these mericlones were tested after 2-3 months of in soil re-establishment and two resulted free from mite-borne virus. The micropropagated shoots, positive in RT-PCR tests, were processed with in vitro thermotherapy or chemotherapy with RIP PAP-II treatment, to verify the possibility to improve the elimination rate of mite borne virus from garlic.