Abstract
A cryopreservation protocol was developed for in vitro shoot tips of Garcinia hombroniana using the vitrification technique. Four critical steps in the technique were investigated, namely preculture, loading, dehydration with Plant Vitrification Solution 2 (PVS2), and unloading. Shoot tips precultured for 48 hr gave significantly higher survival (75 %) compared to 24 hr preculture (50 %) after cryopreservation. Treatment with 1 M glycerol plus 0.4 M sucrose as a loading solution gave higher survival (45.83 %) compared to the other treatments (0.4 M sucrose + 2 M glycerol; 0.4 M sucrose). Shoot tips dehydrated with PVS2 for 25 min gave the highest survival after immersion in liquid nitrogen. Stepwise PVS2 treatment for 15 min with 50 % PVS2 followed by 10 min with 100 % PVS2 solution improved survival of the shoot tips after cryopreservation (41.67 %). Murashige and Skoog medium with 0.4 M sucrose gave significantly higher survival (66.67 %) than MS with 1.2 M sucrose (25 %) as an unloading solution. Water content was shown to decrease throughout the whole vitrification steps from 6.83 ± 1.66 g g-1 dw for fresh shoot tips down to 2.93 ± 0.28 g g-1 dw after PVS2 treatment. Further study on each step including recovery medium is required to improve the survival. Nevertheless, the present study showed the potential of using the vitrification technique for cryopreservation of G. hombroniana.
Highlights
Garcinia hombroniana Pierre (Clusiaceae) is a tropical tree native to Malaysia, Thailand, Borneo and found in the islands of Andaman and Nicobar (Abraham et al, 2008; Nazre, 2010)
Preliminary test on Plant Vitrification Solution 2 (PVS2) exposure: In this experiment, precultured shoot tips were dehydrated with 100 % PVS2 at 0 °C without liquid nitrogen (LN) exposure to test the ability of shoot tips to withstand PVS2 for extended periods of time
There was no survival of LN-exposed shoot tips without PVS2 dehydration
Summary
Garcinia hombroniana Pierre (Clusiaceae) is a tropical tree native to Malaysia, Thailand, Borneo and found in the islands of Andaman and Nicobar (Abraham et al, 2008; Nazre, 2010). Vitrification is the most commonly used technique in cryopreservation This is a simple and reliable technique which allows much more selection of plant materials to be used for cryopreservation (Sakai & Engelmann, 2007). For this technique there are no special equipment needed and the procedure is fast as concentrated solutions are used which shorten. Studies on cryopreservation of G. hombroniana have not been reported far Since this species’ seed is relatively large in size and the availability of seeds depends on the fruiting season, in vitro seed cultures were used for shoot proliferation and shoot tips from these cultures were used as the starting materials for cryopreservation. Suitable preculture duration, loading solution, dehydration period and unloading solution were studied in order to obtain successful cryopreservation of this species
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