In vitro propagation and cryopreservation of Thuja koraiensis Nakai via somatic embryogenesis
- 78 Downloads
Korean arbor vitae (KAV; Thuja koraiensis Nakai) is a critically endangered coniferous tree in Korea. Here, we report the somatic embryogenesis (SE) and cryopreservation system that can be used for micropropagation of KAV and long-term storage of KAV cultures. To induce SE in KAV, the influence of the developmental stage of zygotic embryos and the effect of basal medium on embryogenesis induction were examined. The developmental stage of zygotic embryos had a significant effect on the embryogenesis induction (P < 0.0001). The highest frequency of embryogenesis induction occurred in megagametophytes with zygotic embryos at precotyledonary (P) and late embryogeny (L1) stage (36%). The highest frequency of embryogenesis induction was obtained on initiation medium containing IM basal salts with 2.2 μM 6-benzylaminopurine and 4.5 μM 2,4-dichlorophenoxyacetic acid (35%). The effect of abscisic acid (ABA) on production of somatic embryos was tested. The highest number of somatic embryos per 50 mg of embryogenic tissue was achieved on maturation medium with levels of 100 μM ABA (24.0 ± 2.4). The effect of cryopreservation treatment to embryogenic tissues on the maturation capacity of somatic embryos was also tested. No significant differences between noncryopreservation and cryopreservation treatment were observed (P = 0.1896), and the highest mean number of somatic embryo per 50 mg of embryogenic tissues was obtained in noncryopreserved cell line (28.17 ± 5.66). Finally, the genetic identities of the plantlets regenerated from non- and cryopreserved embryogenic cell lines were verified and there was no genetic variation in the regenerated plantlets from cryostored embryogenic cell lines. This study is the first report on SE and the successful cryopreservation of embryogenic culture of the genus Thuja.
KeywordsCupressaceae Endangered conifer Embryogenic tissue Cryostorage Genetic fidelity
This research was carried out with the support of ‘R&D Program for Forest Science Technology (Project No. FTIS 2018131B10-1820-BB01) provided by Korea Forest Service (Korea Forestry Promotion Institute). We would like to thank the National Park Research Institute of Korea and Korea National Arboretum. We also thank to Dr. Hyo-In Lim (Korea Forest Research Institute) for the assistance.
YEC and CHA designed all experiments and wrote all drafts of the manuscript. CHA conducted the experiments and data analysis. KH carried out cytological analysis. HSP collected KAV cones for culture initiation and provided some photos.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- Ahn CH, Kim YS, Lim S, Yi JS, Choi YE (2011) Random amplified polymorphic DNA (RAPD) analysis and RAPD-derived sequence characterized amplified regions (SCAR) marker development to identify Chinese and Korean ginseng. J Med Plant Res 5:4487–4492Google Scholar
- Aronen TS, Krajnakova J, Häggman HM, Ryynänen LA (1999) Genetic fidelity of cryopreserved embryogenic cultures of open-pollinated Abies cephalonica. Plant Sci 142:163–172Google Scholar
- DeVerno LL, Park YS, Bonga JM, Barrett JD (1999) Somaclonal variation in cryopreserved embryogenic clones of white spruce [Picea glauca (Moench) Voss.]. Plant Cell Rep 18:948–953Google Scholar
- IUCN (2011) IUCN red list categories and criteria. Version 3.1. IUCN Species Survival Commission. IUCN, Gland, Switzerland and Cambridge, U. K.Google Scholar
- Klimaszewska K, Noceda C, Pelletier G, Label P, Rodriguez R, Lelu-Walter M (2009) Biological characterization of young and aged embryogenic cultures of Pinus pinaster (Ait.). In Vitro Cell Dev Biol-Plant 45:20–33Google Scholar
- Ma X, Bucalo K, Determann RO, Cruse-Sanders JM, Pullman GS (2012) Somatic embryogenesis, plant regeneration, and cryopreservation for Torreya taxifolia, a highly endangered coniferous species. In Vitro Cell Dev Biol-Plant 48:324–334Google Scholar
- Mikuła A, Olas M, Sliwinska E, Rybczyński JJ (2008) Cryopreservation by encapsulation of Gentiana spp. cell suspensions maintains regrowth, embryogenic competence and DNA content. Cryo-Letters 29:409–418Google Scholar
- Nour KA, Thorpe TA (1993) In vitro shoot multiplication of eastern white cedar (Thuja occidentalis). In Vitro Cell Dev Biol-Plant 29:65–71Google Scholar
- Ramarosandratana A, Harvengt L, Bouvet A, Calvayrac R, Paques M (2001) Effects of carbohydrate source, polyethylene glycol and gellan gum concentration on embryonal suspensor mass (ESM) proliferation and maturation of maritime pine somatic embryos. In Vitro Cell Dev Biol-Plant 37:29–34Google Scholar
- Song JH, Lee JJ, Koo YB, Lee KY, Han SD, Yang BH (2006) Propagation by cutting method of Korea rare endemic Thuja koraiensis Nak. J Korean For Soc 95:393–397Google Scholar