AOBPreview published online on March 24, 2006
Annals of Botany, doi:10.1093/aob/mcl054
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1 Faculty of Agriculture, Meiji University, Higashi-mita, Tama-ku, Kawasaki, 214-8571, Japan; Genetic Diversity Department, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, 305-8602, Japan
* To whom correspondence should be addressed.
• 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) 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.
Received February 16, 2005
Revised March 8, 2005
Accepted February 1, 2006
Article
Physiological Changes in Gentian Axillary Buds During Two-step Preculturing with Sucrose that Conferred High Levels of Tolerance to Desiccation and Cryopreservation
MITSUTERU SUZUKI 1 
,
MASAYA ISHIKAWA 2 * ,
HITOSHI OKUDA 3,
KATSUJI NODA 4,
TADASHI KISHIMOTO 2,
TOSHIHIDE NAKAMURA 2,
ISAO OGIWARA 5,
ISAO SHIMURA 5,
and
TOMOYA AKIHAMA 6
2 Genetic Diversity Department, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, 305-8602, Japan
3 Department of Citrus Research, National Institute of Fruit Tree Science, Okitsu, Shimizu, 424-0292, Japan
4 Agricultural Science, Gifu University, Yanagido 1-1, Gifu, 501-1112, Japan
5 Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, 183-8509, Japan
6 Faculty of Agriculture, Meiji University, Higashi-mita, Tama-ku, Kawasaki, 214-8571, Japan
MASAYA ISHIKAWA, E-mail: isikawam{at}affrc.go.jp
Abstract These two authors contributed equally to the work.
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