Development of an encapsulation-vitrification protocol for Rubia akane (nakai) hairy roots: a comparison with non-encapsulation.
BACKGROUND
A comparison of different cryopreservation techniques should be based on the characteristics of both the methodology and the material in question using an optimized procedure.


OBJECTIVE
This study aimed at developing an encapsulation-vitrification procedure for hairy roots of Rubia akane using alternative loading and vitrification solutions, based on the existing optimized droplet-vitrification procedure.


METHODS
Encapsulated roots were first precultured in liquid medium with 10% sucrose for 3 days, then with 17.5 % sucrose for 1 day, after which they were osmoprotected with solution C6-40 % (20 % glycerol + 20 % sucrose) for 50 min, cryoprotected with solution A3-90 % (37.5 % glycerol + 15 % DMSO + 15 % EG + 22.5 % sucrose, w/v) on ice for 40 min, cooled and warmed in 2 ml cryovials, and unloaded in 35% sucrose solution for 60 min.


RESULTS
Through the application of this procedure to aged-clustered roots, up to 97.5 % post-cryopreservation regeneration was observed. In our previous study, droplet-vitrification of hairy roots of R. akane resulted in 83.8 % post-rewarming regeneration following preculture with 10 % sucrose for 2 days and 17.5 % sucrose for 4-5 h, and osmoprotection with solution C4-35 % (17.5 % glycerol + 17.5 % sucrose) for 30 min, and cryoprotection with solution A3-70 % (29.2 % glycerol + 11.7 % DMSO + 11.7% EG + 17.4% sucrose, w/v) on ice for 20 min. In the present study, higher post-cryopreservation regeneration was observed by using a higher concentration of vitrification solution (A3-70 % → A3-90 %, B5-80 % → B1-100 %) and/or a longer cryoprotection duration (A3-70 % at room temperature (RT) for 8 min → 15-30 min, on ice for 20 min → 40-80 min; B5-80 % for 15 min → 30-60 min).


CONCLUSIONS
Even though encapsulation provided some degree of protection from the cytotoxicity of vitrification solutions to cytotoxicity-sensitive R. akane hairy roots, an overall higher post-cryopreservation regrowth was obtained using the droplet-vitrification procedure under optimized conditions. This result implies that this sensitive material was not sufficiently cryoprotected, and thus, rapid cooling and warming using foil strips was more efficient than cryopreservation of encapsulated samples.
Species Restoration Center, Korea National Park Service, Gurye, Korea.
D-J
DJ
Shin
Department of Well-being Resources, Sunchon National University, Suncheon, Korea.
H-E
HE
Lee
National Institute of Biological Resources, 42 Nanji-ro, Seo-gu, Incheon, Korea.
C-H
CH
Bae
Division of Plant Science and Resources, Chungnam National University, Daejeon, Korea.
S-U
SU
Park
Species Restoration Center, Korea National Park Service, Gurye, Korea.
H-N
HN
Kang
Department of Well-being Resources, Sunchon National University, Suncheon, Korea. cryohkim@sunchon.ac.kr.
H-H
HH
Kim
D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000378:metabolism*; D009997:Osmotic Pressure / Q000187:drug effects; D018517:Plant Roots / Q000187:drug effects / Q000254:growth & development / Q000502:physiology*; D045470:Rubia / Q000187:drug effects / Q000254:growth & development / Q000502:physiology*; D058989:Vitrification
D003451:Cryoprotective Agents