Cryopreservation of winter-dormant mulberry buds using two-step freezing
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초록
Genetic resources of mulberry trees are commonly preserved as trophosomes, which are vulnerable to environmental factors, such as natural disasters, diseases, and pests. This study establishes a basic protocol for ultra-low temperature cryopreservation of mulberry trees using a two-step freezing process. The procedure was established using the ‘‘Daeshim’’ variety and then tested on genetic resources from 24 other mulberry varieties. Samples were first dried to a moisture content of 33–43% in a low-temperature forced-air chamber at −5 °C, then slowly frozen from −5 °C to −20 °C, and preserved in liquid nitrogen (−196 °C). To determine the regeneration rate, isolated dormant buds were inoculated into MS basal medium, and grown shoots were grafted onto 1-year-old rootstock via chip budding and then cultured. After freezing in liquid nitrogen, the ‘‘Daeshim’’ variety exhibited a survival and regeneration rate of more than 70% and 50%, respectively. Applying the two-step freezing process to genetic resources from 24 mulberry species yielded average survival and regeneration rates of 85.3% and 75.5%, respectively. Morus alba showed survival and regeneration rates of 100%, confirming the efficacy of the two-step freezing method. These results indicate the high feasibility of ultra-low-temperature cryopreservation through two-step freezing of dormant buds from mulberry genetic resources. Additional research is required into the variations in regeneration rates with freezing period in liquid nitrogen.
Abstract
Genetic resources of mulberry trees are commonly preserved as trophosomes, which are vulnerable to environmental factors, such as natural disasters, diseases, and pests. This study establishes a basic protocol for ultra-low temperature cryopreservation of mulberry trees using a two-step freezing process. The procedure was established using the ‘‘Daeshim’’ variety and then tested on genetic resources from 24 other mulberry varieties. Samples were first dried to a moisture content of 33–43% in a low-temperature forced-air chamber at −5 °C, then slowly frozen from −5 °C to −20 °C, and preserved in liquid nitrogen (−196 °C). To determine the regeneration rate, isolated dormant buds were inoculated into MS basal medium, and grown shoots were grafted onto 1-year-old rootstock via chip budding and then cultured. After freezing in liquid nitrogen, the ‘‘Daeshim’’ variety exhibited a survival and regeneration rate of more than 70% and 50%, respectively. Applying the two-step freezing process to genetic resources from 24 mulberry species yielded average survival and regeneration rates of 85.3% and 75.5%, respectively. Morus alba showed survival and regeneration rates of 100%, confirming the efficacy of the two-step freezing method. These results indicate the high feasibility of ultra-low-temperature cryopreservation through two-step freezing of dormant buds from mulberry genetic resources. Additional research is required into the variations in regeneration rates with freezing period in liquid nitrogen.
참고문헌 (23)
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