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Jordan University of Science and Technology (JUST) 2004

In Vitro Preservation of Date Palm Phoenix Dactylifera

وليد سليمان اصبيح الخوالده

Titre : In Vitro Preservation of Date Palm Phoenix Dactylifera

Auteur : وليد سليمان اصبيح الخوالده

Université de soutenance : Jordan University of Science and Technology (JUST)

Grade : Master of Science (MS) 2004

Date palm is one of the most important fruit trees all over the world, for its valuable benefits. In the current study, two in vitro preservation methods were evaluated : short term slow growth preservation (using different osmotic agents and reduced temperature), and cryopreservation (long term preservation) via encapsulation-dehydration, vitrification and encapsulation-vitrification. Embryogenic calli from in vitro grown plantlets were used for slow growth and cryopreservation. Preservation of calli on a medium containing 0.1 or 0.18 M sucrose or 0.16 M mannitol or 0.16 M sorbitol under 16 h of light was able to decrease the growth and maintain explant quality for 12 weeks. Embryogenic calli preservation at 24 ?C was also able to decrease the growth and to maintain the explants in a better condition for 12 weeks. Calli were precultured on various concentrations of sucrose, mannitol or sorbitol for 1,2 or 3 days, to optimize the concentration of osmoticum in preculture medium and preculture duration before cryopreservation. A complete survival was obtained after preculture of the calli with 0.0 and 0.1 M sucrose for 1, 2 or 3 days. In encapsulation-dehydration, the effect of sucrose concentration and dehydration period on survival and regrowth of encapsulated calli were studied. The greatest survival (93.3-100%) and (73.3-80%) regrowth rates were obtained when encapsulated unfrozen calli were pretreated with 0.1 or 0.3 M sucrose, respectively, for 2 days with or without further air dehydration for 2 h. After cryopreservation, high survival (80%) was obtained when calli were pretreated with 0.3 sucrose for 2 days followed by 2 h dehydration where the beads attained 55.4% moisture content. While the highest regrowth (33.3-40%) was obtained with 0.1 M sucrose after 2 or 4 h of dehydration or with 0.3 M sucrose after 2 h of dehydration, or with 0.5 M sucrose after 4 h of dehydration. Viability of calli decreased with increased concentration and dehydration period. Encapsulated calli were also dehydrated with silica gel for various dehydration periods (0, 1, 2, 3 or 4 h) prior to freezing. Dehydration of encapsulated calli with silica gel for 1 or 2 h resulted in maximum rates of 80% survival and 60-66.7% regrowth, respectively. In vitrification, the effect of PVS2 concentration on the viability of calli before and after cryopreservation was examined. Direct exposure of calli to 100% PVS2 decreased the viability of calli. High survival (100%) and 66.7% regrowth rates were achieved with four- step dehydration, using PVS2 at 25 ?C for 20 min prior to freezing. The effect of fourteen different cryoprotectants on survival of calli following cryopreservation was also tested. The use of 2 M glycerol plus 0.4 M sucrose resulted in 86.7% survival rate. Moreover, the effect of combination of three different cryoprotectants and three different vitrification solutions on viability of calli was tested. Cryoprotection by using 1.0 M sucrose plus 15% DMSO and dehydration with 2.0 M glycerol plus 0.4 M sucrose or 0.5 M sucrose plus 10% DMSO produced 60-66.7% survival rates after freezing, respectively. The influence of duration of exposure of calli to 2 M glycerol plus 0.4 M sucrose before dehydration with a highly concentrated PVS2 solution on viability of calli was also experimented. Cryoprotection of calli for 10 or 20 min by using 2 M glycerol plus 0.4 M sucrose produce 60-66.7% survival and 40-46.7% regrowth rates, respectively. Increasing duration of exposure to the cryoprotectant solution decreased the viability of calli. In encapsulation-vitrification, the effect of dehydration of encapsulated calli with 100% PVS2 for various durations of dehydration prior to freezing was studied. Dehydration of encapsulated and cryoprotected calli with 100% PVS2 at 25 ?C for 30 to 60 min resulted in 73.3-80% survival and 60-73.3% regrowth rates, respectively. However, further studies should be initiated to improve slow growth conditions and regrowth of the survived calli and to study genetic stability after cryopreservation.


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