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Title: Recovery patterns, histological observations and genetic integrity in Malus shoot tips cryopreserved using droplet vitrification and encapsulation-dehydration procedures

Author
item LI, BAI-QUAN - Northwest Agricultural & Forestry University
item FENG, CHAO-HONG - Northwest Agricultural & Forestry University
item HU, LING-YUN - Northwest Agricultural & Forestry University
item WANG, MIN-RUI - Northwest Agricultural & Forestry University
item Volk, Gayle
item WANG, QIAOCHUN - Northwest Agricultural & Forestry University

Submitted to: Journal of Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/25/2015
Publication Date: 9/30/2015
Citation: Li, B., Feng, C., Hu, L., Wang, M., Volk, G.M., Wang, Q. 2015. Recovery patterns, histological observations and genetic integrity in Malus shoot tips cryopreserved using droplet vitrification and encapsulation-dehydration procedures. Journal of Biotechnology. 214:182-191.

Interpretive Summary: Apple (Malus) genetic resources are maintained in genebanks worldwide. Apple cultivars are propagated by grafting, and thus are maintained in field collections rather than as seeds. Field collections are expensive to maintain and are at risk of climatic and pathogen threats. In the USDA, most of the apple cultivar collection is securely backed-up as cryopreserved dormant buds. However, due to their location and/or genetic composition, some apple accessions cannot be conserved as dormant buds in liquid nitrogen. This manuscript describes two methods for cryopreserving shoot tips excised from in vitro-grown plants. The two methods resulted in high levels of survival across several species. Observations of cellular damage revealed a correlation between shoot tip survival and the percentage and types of cells that survived. In addition, ploidy and DNA assessments revealed no observed changes in the genetic composition in plants recovered from shoot tips after liquid nitrogen exposure.

Technical Abstract: A droplet-vitrification procedure is described for cryopreservation of Malus shoot tips. Survival patterns, recovery types, histological observations, and genetic integrity were compared for Malus shoot tips cryopreserved using this droplet-vitrification procedure and an encapsulation-dehydration procedure that was previously reported by us. In both procedures, three types of shoot tip recovery were observed following cryopreservation: callus formation without shoot regrowth, leaf formation without shoot regrowth, and shoot regrowth. Three categories of histological observations were also identified in cross-sections of shoot tips recovered after cryopreservation using the two cryogenic procedures. In category 1, almost all of the cells (94-95%) in the apical dome (AD) were damaged or killed and only some cells (30-32%) in the leaf primordia (LP) survived. In category 2, a few cells (18-20%) in the AD and some cells (30-31%) in the LP survived. In category 3, the majority of cells (60-62%) in the AD and some cells (30-33%) in the LP survived. These data suggest that shoot regrowth is correlated to the presence of a majority of surviving cells in the AD after liquid nitrogen exposure. No polymorphic bands were detected by inter-simple sequence repeats or by random amplified polymorphic DNA assessments, and ploidy levels analyzed by flow cytometry were unchanged when plants recovered after cryoexposure were compared to controls. After cryoexposure, mean shoot regrowth levels were higher in the encapsulation-dehydration method (61%) than in the droplet-vitrification method (48%), suggesting the former may be preferred for routine cryobanking applications for Malus shoot tips. The methods appear to be robust since seven genotypes representing four Malus species and one hybrid recovered shoots after both the encapsulation-dehydration and droplet-vitrification procedures were implemented.