Studies On Banana Bunchy Top Virus Elimination By Cryopreservation

Banana bunchy top virus (BBTV) is one of diseases in banana. Popularizing virus-free seedling is an effective way of controlling virus. Shoot-tip culture and heat-treatment are the commonly-used ways. Cryopreservation could not only preserve germplasm but also eliminate virus.This study was carried on Banana cultivar[Musa acuminaxe (AAA group) Dwraf Cavendish cv. Baxi], infected by BBTV. The theory was studied of cryopreservation for the elimination of BBTV from banana. The main results are as follows:1. The explants BBTV-infected were sterilized by 0.1 % HgCl₂ for 12～14 min. The sterilized buds were proliferated preferably on the medium containing MS + 6-BA 4.0 mg/1 + 0.4 NAA mg/L and eugenic on the medium containing MS + 6-BA 2.0 mg/L + NAA 0.2 mg/L.2. Conditions for shoot-tip culture were optimized. When medium containing 6-BA 0.5mg/L was liquid and it was dark culture, rotating 110 ring /min, healthy shoot-tips elongated 0.76 cm after 7 days and proliferation rate was 1.9-fold after 1 month. Meanwhile, shoot-tips elongated 0.65 cm with solid medium and dark culture, proliferation rate was 2.7-fold. The latter method was more economical than the former.3. The essence of vitrifiable cryopreservation was the virus-eliminating of shoot-tip culture. The structure of the cryopreservation shoot-tips was observed by light microscopy. The result showed that cryopreservation acted as a micro-scalpelit kept alive few layers of meristematic but killed the more hydrated parenchymatic cells. Cryopreservation living rate was high, because meristematic cells contained thicker Cytoplasm and lower free water. The meristematic cells carried no- or low-virus. That was why free-virus plant rate had dramatically increased.4. Detection of BBTV by Polymerase Chain Reaction (PCR). Primer up: ATT ACT CGA GCT CGG GAC GGG ACA T (65.3℃); Primer down: TAT ACT CGA GGG GTA ATA ATA TAC CCC(60.5°C). PCR components: 2μl 10·PCR buffer; 0.6μl Taq DNA polymerase (2 U/μl); 0.4μl dNTP (10 mM); 0.6μl Primer (10 mM); 2μl Template DNA; 13.8μl ddH₂O; 20μl in total. Procedures were as follows: 94℃, 3 min; 94μ, 1 min, 54℃, 30 s, 72℃, 30 s; 35cycles, 72℃, 10 min, 12℃hold.5. Conventional shoot-tip culture resulted in 26.7% BBTV-free plants while cryopreservation treatment resulted in 60.6% free plants.