Cryopreservation in vitro of blackberry Cacanska Bestrna shoot tips by encapsulation dehydration
2011
Ružić, Đ., Fruit Research Institute, Čačak (Serbia) | Vujović, T., Fruit Research Institute, Čačak (Serbia)
Over the most recent period, tissue culture group of the Fruit Research Institute (from Cacak, Serbia) has initiated the work on application of cryopreservation techniques for the long-term conservation of temperate fruit species, valuable supplement to the traditional germplasm preservation. In the present study, in vitro grown shoot tips of blackberry (Rubus fruticosus L.) Cacanska Bestrna were tested for regrowth after cryopreservation using slightly modified encapsulation dehydration method described by Dereuddre et al. (1990). Apical shoot tips 2-3 mm length were encapsulated in alginate beads composed of 3, 5 and 10% (w/v) alginic acid sodium salt in calcium-free liquid MS medium (Murashige and Skoog, 1962) supplemented with 1 mg |-1 BA, 0.1 mg |- 1 IBA and o.1 mg |-1 GA3. Polymerization was done in liquid MS medium with 100 mM CaCl2 for 30 min at room temperature. Encapsulated shoot tips were pre-treated with liquid MS medium with 0.75 or 1 M sucrose for 24 h in growth room and dehydrated for 4 and 8 h to 29% and 20% moisture content (fresh weight basis) before rapid immersion in liquid nitrogen (LN) for 1 h. Upon thawing performed by placing the cryovials in the air currant of the laminar airflow cabinet for 2 min the beads were directly transferred on a regrowth medium. Explants showing normal development (expansion of new leaves and/or development of a small shootlets) 31 days after freezing were considered to be regrowing. The osmotic dehydration in 1 M sucrose followed by 8 h of desiccation gave the highest survival rate (22%) and regrowth (16.7%) after freezing in LN for explants encapsulated in 5% alginate beads. Cryopreserved shoot tips multiplied in the first subculture and had normal morphology with similar multiplication capacity in comparison with non- cryopreserved shoots.
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