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Annals of Botany 89: 391-400, 2002
© 2002 Annals of Botany Company

In vivo Characterization of the Effects of Abscisic Acid and Drying Protocols Associated with the Acquisition of Desiccation Tolerance in Alfalfa (Medicago sativa L.) Somatic Embryos

LEKHA SREEDHAR{dagger},1, WILLEM F. WOLKERS{ddagger},2, FOLKERT A. HOEKSTRA*,2 and J. DEREK BEWLEY1

1Department of Botany, University of Guelph, Guelph, Ontario, Canada N1G 2W1 and 2Department of Plant Sciences, Laboratory of Plant Physiology, Wageningen University, Wageningen, The Netherlands

* For correspondence. Fax +31 317 484740, e-mail folkert.hoekstra{at}pph.dpw.wau.nl
{dagger} Present address: Cell Biology & Biophysics, School of Biological Sciences, University of Missouri-Kansas City, MO 64110, USA.
{ddagger} Present address: Section of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA.

Received: 9 April 2001; Returned for revision: 10 November 2001; Accepted: 15 December 2001.

Although somatic embryos of alfalfa (Medicago sativa L.) had acquired some tolerance to desiccation at the cotyledonary stage of development (22 d after plating), additional culturing in 20 µM abscisic acid (ABA) for 8 d induced greater desiccation tolerance, as determined by increased germination. Compared with fast drying, slow drying of the ABA-treated embryos improved desiccation tolerance. However, slow drying of non-ABA-treated embryos led to the complete loss of germination capacity, while some fast-dried embryos survived. An electron paramagnetic resonance spin probe technique and in vivo Fourier transform infrared microspectroscopy revealed that cellular membrane integrity and {alpha}-helical protein secondary structure were maintained during drying in embryos cultured in media enriched with 20 µM ABA, but not in embryos cultured in the absence of ABA. Slow-dried, non-ABA-treated embryos had low oligosaccharide to sucrose ratios, an increased proportion of ß-sheet protein secondary structures and broad membrane phase transitions extending over a temperature range of more than 60 °C, suggestive of irreversible phase separations. The spin probe study showed evidence of imbibitional damage, which could be alleviated by prehydration in humid air. These observations emphasize the importance of appropriate drying and prehydration protocols for the survival and storage of somatic embryos. It is suggested that ABA also plays a role in suppressing metabolism, thus increasing the level of desiccation tolerance; this is particularly evident under stressful conditions such as slow drying.

Key words: Abscisic acid, alfalfa (Medicago sativa L.), desiccation tolerance, electron paramagnetic resonance spectroscopy, Fourier transform IR spectroscopy, germination, membrane integrity, protein secondary profile, somatic embryos, sugars.


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