Application of cryobanking for Platycodon grandiflorum in vitro axillary buds using cryo-plate methods
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BIOTECHNOLOGY
Application of cryobanking for Platycodon grandiflorum in vitro axillary buds using cryo-plate methods Toshikazu Matsumoto 1 & Daisuke Tanaka 2 & Kayo Yoshimatsu 3 & Noriaki Kawano 3 & Nobuo Kawahara 3 & Shinya Maki 4 & Shin-ichi Yamamoto 2 & Takao Niino 5 Received: 27 May 2020 / Accepted: 19 August 2020 / Editor: Barbara Reed # The Society for In Vitro Biology 2020
Abstract The application of the V cryo-plate method using either PVS2 or PVS3 and the D cryo-plate method to Platycodon grandiflorum in vitro axillary buds was investigated. Precultured buds (1 mm) were attached to cryo-plates using calcium alginate gel. Then, osmoprotection treatment (2 M glycerol and 1 M sucrose) was performed by immersing the cryo-plates for 30 min at 25°C. The procedures for each cryogenic method were as follows: in the V cryo-plate method, the buds on cryo-plates were exposed to PVS2 for 40 min (highest regrowth 71.1%) or PVS3 for 50 min (highest regrowth 82.2%). In the D cryo-plate method, the buds on the cryo-plates were dehydrated under the air current of a laminar air flow cabinet for 60 min (highest regrowth 80.0%) at 25°C. Then, the cryo-plates were plunged directly into liquid nitrogen. After cryopreservation, buds on the cryo-plates were rewarmed and unloaded by immersion in 1 M sucrose solution for 15 min at 25°C for subsequent plant regeneration. The average regrowth seen on the V cryo-plates exposed to PVS3 and in the D cryo-plate method was higher than that of the V cryo-plates exposed to PVS2. The regrowth after cryopreservation in the V cryo-plate method with PVS2 was not stable. Thus, the V cryoplate method with PVS3 and the D cryo-plate method are considered to be practical cryopreservation methods for P. grandiflorum germplasm. Keywords D cryo-plate . Genebank . PVS2 . PVS3 . V cryo-plate
Introduction Platycodon grandiflorum A. DC. belongs to the Campanulaceae family and is called ‘Kikyo’ in Japanese, ‘Doraji’ in Korean and ‘Jieging’ in Chinese (Lee et al. 2004). This plant is used in medicines and also as a food in Korea and northern China (Fu et al. 2006). The roots have * Toshikazu Matsumoto [email protected] 1
Faculty of Life and Environmental Sciences, Shimane University, Matsue, Shimane, Japan
2
Genetic Resources Center, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan
3
Tsukuba Division, Research Center for Medicinal Plant Resources, National Institutes of Biomedical Innovation, Health and Nutrition, Tsukuba, Ibaraki, Japan
4
Department of Science and Technology Innovation, Nagaoka University of Technology, Nagaoka, Niigata, Japan
5
Formary University of Tsukuba, Tsukuba, Japan
been used as a food material, and in traditional eastern medicines as expectorants and antitussives to treat coughs, excessive phlegm, and sore throats, among other ailments (Zhang et al. 2015). The prescription of Kampo medicines in Japan relies on the supplies obtained from wild plants, with almost all the supply of crude drugs dependent on imports. A suitable gene
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