Liquid nitrogen (LN) storage has been recommended for the long-term conservation of plants with seeds that do not survive traditional seed banking, such as short-lived or recalcitrant seeds, but there are few reports confirming its effectiveness for these species. In this study, embryonic axes of Juglans nigra (short-lived), Aesculus hippocastanum and A. glabra (recalcitrant) stored for 11-23 yrs in LN were thawed, cultured in vitro, and evaluated for survival. Excised axes were originally dried under the air flow of a laminar flow hood to moisture levels of 5% (J. nigra) to 30% (Aesculus spp.), transferred to cryovials, and cryopreserved by direct immersion of the cryovials in LN. At the time of storage, survival of a cryopreserved control was evaluated by culturing the axes on MS medium with 2 mg/L each of BAP and IAPhe, but studies with freshly harvested axes and axes removed after 9.5 yrs indicated that growth regulators were not needed, and in this study, samples were cultured without hormones. Embryos were judged to be alive when swelling or growth had occurred after two weeks of culture. Axes of J. nigra stored for 23 years in LN maintained the high viability (>80%) that had been observed both initially and after 9.5 years. Similarly, embryos stored for 11 yrs showed high viability (60-100%), depending on the accession. The normal-appearing plantlets from the 23 year-old axes were successfully acclimatized to soil, although conditions in the lab/greenhouse did not support continued growth of all but one of the plantlets. Axes of A. glabra showed a decline in viability to 33% after 23 years in storage, a decline from 75% after 9.5 yrs. However, viability of A. hippocastanum axes remained high (90%) after 23 years in LN. Surviving Aesculus axes did not produce plants, but did produce somatic embryos or axillary shoots. These results demonstrate that LN storage can preserve embryo axes of J. nigra and Aesculus spp., for at least two decades, providing a workable conservation tool for these species that cannot survive in traditional seed banks. (This work supported in part by IMLS grant # LG-25-12-0595).
In Vitro Recovery of Embryonic Axes of Temperate Trees after 11-23 Years of Storage in Liquid Nitrogen
BALLESTEROS BARGUES, DANIEL;
2015-01-01
Abstract
Liquid nitrogen (LN) storage has been recommended for the long-term conservation of plants with seeds that do not survive traditional seed banking, such as short-lived or recalcitrant seeds, but there are few reports confirming its effectiveness for these species. In this study, embryonic axes of Juglans nigra (short-lived), Aesculus hippocastanum and A. glabra (recalcitrant) stored for 11-23 yrs in LN were thawed, cultured in vitro, and evaluated for survival. Excised axes were originally dried under the air flow of a laminar flow hood to moisture levels of 5% (J. nigra) to 30% (Aesculus spp.), transferred to cryovials, and cryopreserved by direct immersion of the cryovials in LN. At the time of storage, survival of a cryopreserved control was evaluated by culturing the axes on MS medium with 2 mg/L each of BAP and IAPhe, but studies with freshly harvested axes and axes removed after 9.5 yrs indicated that growth regulators were not needed, and in this study, samples were cultured without hormones. Embryos were judged to be alive when swelling or growth had occurred after two weeks of culture. Axes of J. nigra stored for 23 years in LN maintained the high viability (>80%) that had been observed both initially and after 9.5 years. Similarly, embryos stored for 11 yrs showed high viability (60-100%), depending on the accession. The normal-appearing plantlets from the 23 year-old axes were successfully acclimatized to soil, although conditions in the lab/greenhouse did not support continued growth of all but one of the plantlets. Axes of A. glabra showed a decline in viability to 33% after 23 years in storage, a decline from 75% after 9.5 yrs. However, viability of A. hippocastanum axes remained high (90%) after 23 years in LN. Surviving Aesculus axes did not produce plants, but did produce somatic embryos or axillary shoots. These results demonstrate that LN storage can preserve embryo axes of J. nigra and Aesculus spp., for at least two decades, providing a workable conservation tool for these species that cannot survive in traditional seed banks. (This work supported in part by IMLS grant # LG-25-12-0595).
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