Study on the cryopreservation of in vitro shoot tips of Musa germplasm by droplet vitrification

Droplet vitrification method for cryopreservation of shoot-tips of AAA and ABB genotypes banana(Musa spp.) were optimized using orthogonal table of the time for loading of cryoprotective solution,dehydrating and recovery.The effects of different cryoprotectors and shoot tip types on cryopreservation were also studied.The appropriate procedure was as follows.Shoot-tip 1.5 to 2.0 mm in length carrying 1 or 2 leaf primordia was excised and then pretreated with loading solution for 120 min at room temperature.After loading,the solution was replaced by ice-cooled vitrification solution(PVS2) for 30 to 50 min.Then the shoot-tip was transferred to a droplet of PVS2 solution on a strip of aluminum foil and plunged into liquid ni-trogen and stayed in liquid nitrogen for at least one hour.The strips were rinsed in unloading solution at room temperature for 15 min.Then shoot-tips were placed onto a piece of sterilized filter paper on top of semi-solid MS medium containing 0.3 mol.L-1 sucrose.After 24 h,the shoot-tips were transferred onto a regeneration medium without filter paper.Using this droplet vitrification protocol,we successfully cryopreserved 15 accessions belonging to 5 different genomic groups of Musa germplasm.The average survival rate was 78.0% for AAA group,57.8% for AAB group,72.1% for ABB group,43.3% for AA group,and 42.2% for M.itinerans.