Cryopreservation of Embryogenic Callus of Dioscorea bulbifera by Vitrification

Authors: Hong, Senrong; Yin, Minghua; Shao, Xinghua; Wang, Aiping; Xu, Weihong

Source: Cryoletters, Volume 30, Number 1, January 2009 , pp. 64-75(12)

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Abstract:

Cryopreservation of callus of Dioscorea bulbifera by vitrification was optimized. Calli of Dioscorea bulbifera were pretreated in liquid Murashige and Skoog (MS) medium supplemented with 2 mg L⁻¹ kinetin (KT), 0.5 mg L⁻¹ NAA, 0.5 mg L⁻¹ 2,4-D and 0.2 M sucrose for 5 d under continuous light (36 μM m⁻² s⁻¹) at 25±1°C. The material was then loaded with 60% vitrification solution (PVS₂) for 20 min at room temperature and dehydrated with 100% PVS2 for 30 min at 0°C. After changing the solution with fresh PVS2, the calli were directly immersed in liquid nitrogen and conserved for 1-360 d. After rapid thawing in a water-bath at 35°C, the calli were washed three times with liquid MS medium supplemented with 2 mg L⁻¹ KT, 0.5 mg L⁻¹ NAA, 0.5 mg L⁻¹ 2, 4-D and 1.2 M sucrose and then transferred onto solid MS medium supplemented with KT 2 mg L⁻¹, NAA 0.5 mg L⁻¹, 0.09 M sucrose and 0.75% (w/v) agar. The cultures were kept in the dark for 2 days prior to exposure to the light (12 h light/dark cycle). The TTC test showed that 80-90% of the calli survived this cryoprocedure and there was a 60-70% regeneration of plantlets from the calli. The regenerated material did not exhibit any morphological variations.

Keywords: DIOSCOREA BULBIFERA; CRYOPRESERVATION; VITRIFICATION; CALLUS

Document Type: Research article

Publication date: 2009-01-01

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CryoLetters is a bimonthly international journal for low temperature sciences, including cryobiology, cryopreservation or vitrification of cells and tissues, chemical and physical aspects of freezing and drying, and studies involving ecology of cold environments, and cold adaptation
The journal publishes original research reports, authoritative reviews, technical developments and commissioned book reviews of studies of the effects produced by low temperatures on a wide variety of scientific and technical processes, or those involving low temperature techniques in the investigation of physical, chemical, biological and ecological problems.

Cryopreservation of Embryogenic Callus of Dioscorea bulbifera by Vitrification

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