Effect of load solution and growth media on cryopreservation by droplet vitrification of Hancornia speciosa shoot tips

Abstract

Hancornia speciosa Gomes is a native fruit species from Brazil which produces recalcitrant seeds and thus, conservation methods must be studied in order to preserve the species. The objectives of this study were: (i) to subject shoot tips to different exposure periods to loading solution and, (ii) test different regeneration culture media for cryopreserved explants. Shoot tips were excised from in vitro established plants and pre-cultured in Woody Plant Medium with 0.3 M sucrose for 24 h. After pre-culture, shoot tips were subjected to different exposure periods to loading solution (20, 60, 90 and 120 min), followed or not by treatment in Plant Vitrification Solution 2 at 0°C for 30 min before being plunged into liquid nitrogen. Thawing was performed in unloading solution and explants were post-cultured on Woody Plant Medium with 0.3 M sucrose for 24 h and subsequently transferred to Woody Plant Medium with 0.09 M sucrose and 2 mg/L 6-benzyladenine. Shoot tips were also excised and pre-cultured, treated with LS following PVS2 and then cryopreserved. Shoot tips were thawed in unloading solution and post-cultured before transfer to: WPM medium with 2 mg/L BA, 20 ppm ascorbic acid and 0.09 M sucrose or WPM with 0.75 mg/L 6-benzyladenine, 0.05 mg/L α-naphthaleneacetic acid, 100 ppm ascorbic acid, 800 ppm polyvinylpyrrolidone and 0.09M sucrose. The use of different exposure periods to loading solution without the treatment in Plant Vitrification Solution 2 did not allow shoot tips survival, while the PVS2 treated shoot tips showed 92% post-thaw survival and 77% regrowth after 30 days culture. Regarding the shoot tips regeneration medium, it was observed that the WPM medium containing BA and NAA significantly increased the shoot tips regrowth. Shoot tips of H. speciose are successfully cryopreserved when treated with Plant Vitrification Solution 2 and the Woody Plant Medium supplemented with 6-benzyladenine and α-naphthaleneacetic acid is effective for shoot tips regeneration after cryopreservation.