Cryopreservation of Shoot Tips of Dendrobium Walter Oumae by Encapsulation/Dehydration

Abstract

In vitro shoot tips of Dendrobium Walter Oumae were cryopreserved using the encapsulation/ dehydration technique. Shoot tips were encapsulated in calcium-alginate before preculture on modified Vacin and Went (1949) agar medium supplemented with 0.3, 0.5 and 0.7 M sucrose for 2 d at 25±2 oC, 37 µmol m-2s-1 for 16 h per d. Encapsulated shoot tips were then dehydrated by incubation in the sterile air flow of a laminar air-flow cabinet for 0-10 h and immediately plunged into liquid nitrogen (LN). After recovering from LN and rapid thawing in a waterbath (40±2 oC), the survival ratio and regrowth were measured by a 2,3,5-triphenyl tetrazolium chloride (TTC) assay and a regrowth culture test, respectively. The highest survival ratio (16.18 mg living cells/100 mg total cells) and regrowth (13.33%) were obtained from encapsulated shoot tips that were previously precultured on 0.3 M sucrose agar medium for 2 d and sufficiently dehydrated for 6-8 h before storing in LN. The regrowth of plantlets was measured from length of shoot tips, number of shoots and roots which did not show any significant difference when air-dehydration and LN were tested. Finally, the regrown shoot tips could develop directly into complete plantlets without protocorms formation and they had normal morphology.