title: Recovery patterns, histological observations and genetic integrity in Malus shoot tips cryopreserved using droplet-vitrification and encapsulation-dehydration procedures.
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 (LPs) survived. In category 2, only a few cells (18-20%) in the AD and some cells (30-31%) in the LPs survived. In category 3, majority of the cells (60-62%) in the AD and some cells (30-33%) in the LPs 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. The droplet-vitrification procedure appears to be robust since seven genotypes representing four Malus species and one hybrid recovered shoots following cryopreservation. Mean shoot regrowth levels of these seven genotypes were 48% in the droplet-vitrification method, which were lower than those (61%) in the encapsulation-dehydration procedure reported in our previous study, suggesting the latter may be preferred for routine cryobanking applications for Malus shoot tips.
authors.affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas, College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, PR China.
authors.forename: Bai-Quan
authors.initials: BQ
authors.lastname: Li
authors.affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas, College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, PR China.
authors.forename: Chao-Hong
authors.initials: CH
authors.lastname: Feng
authors.affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas, College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, PR China.
authors.forename: Min-Rui
authors.initials: MR
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas, College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, PR China.
authors.forename: Ling-Yun
authors.initials: LY
authors.lastname: Hu
authors.affiliation: USDA-ARS National Center for Genetic Resources Preservation, 1111 S. Mason St, Fort Collins, CO 80521, USA.
authors.forename: Gayle
authors.initials: G
authors.lastname: Volk
authors.affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas, College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, PR China. Electronic address: qiaochunwang@nwsuaf.edu.cn.
authors.forename: Qiao-Chun
authors.initials: QC
authors.lastname: Wang
mesh_terms: D015925:Cryopreservation / Q000379:methods*; D018744:DNA, Plant / Q000032:analysis / Q000235:genetics; D003890:Desiccation / Q000379:methods*; D027845:Malus / Q000166:cytology* / Q000235:genetics / Q000502:physiology*; D018520:Plant Shoots / Q000166:cytology* / Q000502:physiology*; D058989:Vitrification
chemical_list: D018744:DNA, Plant
keywords: Droplet-vitrification; Encapsulation-dehydration; Genetic stability; Histological observation; Malus; Shoot tips