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In vitro germplasm conservation of elite Stevia rebaudiana Bertoni

Lata, H., Chandra, S., Wang, Y. H., ElSohly, M. A., Khan, I. A.
Acta horticulturae 2014 no.1039 pp. 303-308
Stevia rebaudiana, artificial seeds, calcium chloride, encapsulation, explants, germplasm conservation, glucose, high performance liquid chromatography, mannitol, plantlets, regrowth, shoots, sodium alginate, sorbitol, stevioside, thidiazuron, tissue culture
Germplasm conservation of elite Stevia rebaudiana Bert. was attempted by using synthetic seed technology and medium with added osmotic agents. Explants of nodal segments containing single axillary bud were excised from in vitro proliferated shoot cultures and encapsulated in high-density sodium alginate (5%) hardened by 50 mM CaCl2. The encapsulated and non-encapsulated nodal segments were stored at 5, 15 and 25°C for 4, 8 and 12 months and monitored for the re-growth and survival frequency under the tissue culture conditions on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ 0.2 mg L-1). Encapsulated nodal segments could be stored at 25°C up to eight months with maximum regrowth ability and survival frequency of 77%. The highest re-growth in non-encapsulated cultures was observed in the explants kept at 25°C without osmotic agents, however, with a lower survival frequency. The effect of osmotic agents dextrose, mannitol and sorbitol on non-encapsulated shoot cultures was also evaluated. A considerable decrease in re-growth and survival was observed in the cultures treated with osmotic agents. Among the cultures treated with osmotic agents, the highest rate of re-growth and survival was observed at 2% sorbitol followed by 2% sorbitol + 2% dextrose. Well-developed plantlets regenerated from encapsulated nodal segments were successfully acclimatized inside the growing room with 87% survival. High-performance liquid chromatography was used to assess the stability in chemical profile and quantification of rebaudioside A and stevioside content of mother plant (MP) and the plants grown through synthetic seeds after 4, 8 and 12 months of storage under slow growth conditions. Our results show no significant differences (p < 0.05) in rebaudioside A and stevioside content between the mother and re-grown plants following in vitro storage.