Physiological Changes in Gentian Axillary Buds During Two-step Preculturing with Sucrose that Conferred High Levels of Tolerance to Desiccation and Cryopreservation
Abstract
• Background and Aims Induction of dehydration tolerance is a key to achieving high survival rates in cryopreservation of plant specimens. It has been reported previously that two-step preculturing with sucrose effectively increased desiccation tolerance in axillary buds of gentian (Gentiana scabra), which allow the buds to survive cryopreservation. This study is aimed at characterizing each step of this preculturing and to elucidate physiological changes induced during this preculturing.
• Methods In standard two-step preculture, excised gentian axillary buds were incubated for 11 d on MS medium with 0·1 m sucrose at 25 °C (first step: mild osmotic stress was given) and the subsequent incubation on MS medium with 0·4 m and 0·7 m sucrose for 1 d each (second step). The levels of abscisic acid (ABA), proline and soluble sugars in gentian buds during the preculture were determined. Effects of various combinations of two-step preculturing and of exogenous ABA and proline were studied.
• Key Results During the first preculture step, there was a transient increase in ABA content peaking on day 4, which declined to a background level at the end of the first and second step preculturing. Proline level increased steadily during the first preculture step and increased further in the second preculture step. Incubating buds with medium containing proline, instead of the two-step preculturing, did not allow them to survive desiccation. Incubating buds with ABA instead of 0·1 m sucrose-preculturing effectively increased desiccation tolerance only when it was followed by the second preculture step. Fluridone, an ABA synthesis inhibitor included in the two-step preculture medium, reduced desiccation tolerance of the buds. The normal first-step preculture increased the levels of soluble sugars 2·4-fold, especially sucrose and raffinose. Buds treated with the second preculture step had greatly increased sucrose levels.
• Conclusions These observations lead to the hypothesis that the first preculture step involves ABA-mediated cellular changes and the second step induces loading of sucrose in the gentian buds.
Axillary buds were precultured in a two-step manner as shown in Fig. 1 before being subjected to desiccation at 25 °C and approx. 45 % RH and subsequently immersed into LN. Ten or more axillary buds were treated for each of three replicates.
In experiment A, the buds were precultured on MS medium (15 g L sucrose) containing the designated concentration of ABA for 12 d at 25 °C (without second-step preculture) prior to desiccation treatments.
In experiment B, the buds were precultured on MS medium (15 g L sucrose) containing the designated concentration of ABA for 11 d at 25 °C and subsequently on ABA-free MS medium containing 0·4 m and 0·7 m sucrose for 1 d each at 25 °C (same as the second preculture step) before desiccation treatment.
In experiment C, ABA (0–10 mg L) was added to the medium of both preculture steps. The buds were precultured on MS media containing ABA and sucrose (0·1 m) for 11 d in the first step and then on media with ABA and sucrose (0·4 and 0·7 m) for 1 d each in the second step at 25 °C prior to desiccation. In all the experiments, the precultured buds were desiccated for 16 h at 25 °C and RH 45 % to 10 % (f. wt basis) water content. The data are the mean ± standard error of triplicates (ten or more buds for each replicate).
Acknowledgments
We thank Dr Duncan A. Vaughan (NIAS) for critically reading the manuscript and Ms A. Oda and H. Nakatani (NIAS) for helping to prepare the figures and maintaining the in vitro gentian cultures. M. Suzuki acknowledges the support of JSPS Fellowships for Young Scientists.