Effect of Preculture, PVS2 and Vitamin C on Survival of Recalcitrant Nephelium Ramboutan-ake Shoot Tips After Cryopreservation by Vitrification

This paper reports the cryopreservation of Nephelium ramboutan-ake shoot tips derived from in vitro shoot multiplication and in vitro seed germination using vitrification. Preculture with either 0.5 M sucrose for 2 days or a combination of 0.3 M sucrose and 0.5 M glycerol for 3 days enhanced dehydration tolerance and resulted in the highest survival of shoot tips; however, none of the shoot tips withstood liquid nitrogen (LN) exposure. The use of a lower temperature (0°C) during exposure to plant vitrification solution (PVS2) led to higher survival of shoot tips, compared to exposure at 25°C. The survival percentage of shoot tips exposed to PVS2 for up to 20 min at 0°C was 83.3%. It was only 53.3% when shoot tips were exposed to PVS2 at 25°C for 5 min. The importance of vitamin C for reducing oxidative stress in shoots tips was demonstrated. The addition of 0.28 mM vitamin C during critical steps of the vitrification process resulted in a high survival (96.67%) without LN exposure, compared to 73.3% for shoot tips not treated with vitamin C. Moreover, 3.3% shoot tips withstood LN exposure when vitamin C was added during the loading step. This result suggests that cryopreservation is possible for this tropical, recalcitrant seeded tree species.