Articles
ENCAPSULATION-DEHYDRATION METHOD ELEVATES EMBRYOGENIC ABILITIES OF GENTIANA KURROO CELL SUSPENSION AND CARRYING ON GENETIC STABILITY OF ITS REGENERANTS AFTER CRYOPRESERVATION
Article number
908_16
Pages
143 – 154
Language
English
Abstract
The aim of this study was to investigate the influence of cryotreatment on somatic embryogenesis efficiency in Gentiana kurroo cell suspension cultures and to assess the quality and uniformity of regenerants obtained in post-thawing culture.
For cryopreservation, the encapsulation-dehydration method was employed.
To investigate the influence of cryotreatment on somatic embryo production, two embryogenic suspensions of G. kurroo being in various morphogenic phases were used.
Proliferating suspension (PS) consisted mostly of intensively dividing cell clumps, whereas embryos-rich suspension (ES) also included numerous aggregates with proembryos and somatic embryos at the globular stage.
Cell aggregates of both suspensions were encapsulated in alginate beads and exposed to a gradually increased sucrose concentration of up to 1.0 mol/L for a 7-day long osmotic desiccation (OD), 5-h of air desiccation (AD) and an immersion in liquid nitrogen (LN). Six weeks after recovery of suspension cultures, the influence of cryotreatment on somatic embryo production was assessed.
An average of 5 and 29 somatic embryos regenerated from the non-treated (control) suspension culture of PS and ES, respectively.
Osmotic dehydration increased the somatic embryogenesis efficiency about 20 and 9 times for PS and ES, respectively.
Further steps of cryprocedure, air desiccation and LN submersion did not significantly affect it.
For the description of regenerants derived from non-crypreserved and cryopreserved tissue, the total nuclear DNA content by flow cytometry and differences on DNA sequence and methylation pattern levels by modified metAFLP analysis were used.
Cryopreservation did not change the genome size.
Results of metAFLP analysis indicated that site methylation changes induced higher variation than DNA sequence mutations.
However, cryopreservation decreased DNA methylation level but DNA sequence pattern was not changed.
For cryopreservation, the encapsulation-dehydration method was employed.
To investigate the influence of cryotreatment on somatic embryo production, two embryogenic suspensions of G. kurroo being in various morphogenic phases were used.
Proliferating suspension (PS) consisted mostly of intensively dividing cell clumps, whereas embryos-rich suspension (ES) also included numerous aggregates with proembryos and somatic embryos at the globular stage.
Cell aggregates of both suspensions were encapsulated in alginate beads and exposed to a gradually increased sucrose concentration of up to 1.0 mol/L for a 7-day long osmotic desiccation (OD), 5-h of air desiccation (AD) and an immersion in liquid nitrogen (LN). Six weeks after recovery of suspension cultures, the influence of cryotreatment on somatic embryo production was assessed.
An average of 5 and 29 somatic embryos regenerated from the non-treated (control) suspension culture of PS and ES, respectively.
Osmotic dehydration increased the somatic embryogenesis efficiency about 20 and 9 times for PS and ES, respectively.
Further steps of cryprocedure, air desiccation and LN submersion did not significantly affect it.
For the description of regenerants derived from non-crypreserved and cryopreserved tissue, the total nuclear DNA content by flow cytometry and differences on DNA sequence and methylation pattern levels by modified metAFLP analysis were used.
Cryopreservation did not change the genome size.
Results of metAFLP analysis indicated that site methylation changes induced higher variation than DNA sequence mutations.
However, cryopreservation decreased DNA methylation level but DNA sequence pattern was not changed.
Authors
A. Mikuła, K. Tomiczak, A. Wójcik, J.J. Rybczyński
Keywords
cryopreservation, gentian, flow cytometry, metAFLP, nuclear DNA content, somatic embryogenesis, regenerants
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