Cryopreservation of pharmaceutically important orchid Dendrobium chrysanthum Wall. ex Lindl. using vitrification based method
- 704 Downloads
Our present study constitutes the successful and efficient protocol for cryopreservation of Dendrobium chrysanthum. D. chrysanthum Wall. ex Lindl. is a pharmaceutically valuable, ornamental epiphytic orchid of temperate and subtropical regions. On account of excellent herbal medicinal value and horticultural importance, D. chrysanthum is becoming rare due to over exploitation. For long-term conservation of this orchid, protocorm-like bodies of D. chrysanthum were excised and used for cryopreservation by encapsulation–vitrification. In this cryogenic procedure, PLBs were initially osmoprotected with a mixture of 0.4 M sucrose and 2 M glycerol, incorporated in the encapsulation matrix (comprising of 3 % (w/v) sodium alginate and 0.1 M CaCl2). Encapsulated protocorm-like bodies (PLBs) were then precultured on MS liquid medium supplemented with different concentrations of sucrose (0.06, 0.3, 0.5, 0.7 M), and loaded in a loading solution (comprised of 2 M glycerol and 0.4 M sucrose) for different duration to make the precultured PLBs tolerant to plant vitrification solution 2 (PVS2). Subsequently, the PLBs were subjected to PVS2 (Sakai et al. 1990) treatment at different time of exposure (minutes) and temperatures (0 °C and 25 °C). Encapsulated–vitrified PLBs were plunged directly into liquid nitrogen and stored for 1 h. Optimum result (survival 63.2 % and regrowth 59.9 %) was obtained when the beads treated with loading solution for 80 min followed by PVS2 treatment for 100 min. Regenerated plants showed normal morphology as that of control plants.
KeywordsDendrobium chrysanthum Protocorm-like bodies A-naphthaleneacetic acid 6-benzylaminopurine Plant vitrification solution 2
Plant vitrification solution 2
The first author is thankful to UGC Centre for Advanced Studies in Botany, North Eastern Hill University (NEHU), Shillong, India for financial support. SAIF, NEHU is highly acknowledged for supply of liquid nitrogen.
- Bajaj YPS (1995) Cryopreservation of plant cell, tissue organ culture for the conservation of germplasm and biodiversity. In: Bajaj YPS (ed) Biotechnology in agriculture and forestry, vol 32., Cryopreservation of plant germplasm ISpringer, Berlin, pp 3–47Google Scholar
- Hirai D, Sakai A (2001) Recovery growth of plants cryopreserved by encapsulation–vitrification. Bull Hokkaido Prefect Agric Exp Station 80:55–64Google Scholar
- Moges AD, Karam NS, Shibli RA (2004) Cryopreservation of African violet (Saintapulia ionantha Wendl.) shoot tips. In Vitro Cell Develop Biol-Plant 40:389–398Google Scholar
- Poobathy R, Nair H, Subramaniam S (2009) Optimisation of encapsulation-dehydration protocol for the orchid hybrid Ascocenda ‘princess mikasa'. Adv Environ Biol 3:69–83Google Scholar
- Pornchuti W, Thammasiri K (2008) Cryopreservation of protocorms of Dendrobium virgineum Rchb.f. Acta Hortic 788:63–68Google Scholar
- Sakai A, Kobayashi S, Oyiyama (1990) Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. Var. brasiliensis Tanka) by vitrification. Plant Cell Rep 9:30–33Google Scholar
- Salaj T, Matusˇıkova′ I, Fra′terova′ L, Pirsˇelova′ B, Salaj J (2011) Regrowth of embryogenic tissues of Pinus nigra following cryopreservation. Plant Cell Tissue Organ Cult 106:55–61Google Scholar
- Shibli RA, Shatnawi MA, Subaih W, Ajlouni MM (2006) In vitro conservation and cryopreservation of plant genetic resources: a review. World J Agri Sci 2:372–382Google Scholar