Recovery, histological observations and genetic integrity in coconut (Cocos nucifera L.) embryogenic calli cryopreserved using encapsulation-dehydration procedure

Encapsulation-dehydration protocol was evaluated for the long-term conservation of a coconut embryogenic calli (EC). Encapsulated EC were pretreated in a sucrose medium (either 0.5 M or 0.75 M) for different durations (24, 48 and 72 h) followed by desiccation in silica gel (8, 12, 16 and 20 h) prior to storage in liquid nitrogen. Survival and regrowth of EC following desiccation alone or desiccation and freezing was recorded after four and eight months respectively. Histological studies and scanning electron microscopy (SEM) were carried out to evaluate the structural changes of cryopreserved samples. Recovered samples from cryopreserved and non-cryopreserved EC were tested for genetic fidelity at eleven Simple Sequence Repeat (SSR) marker loci. Highest survival (45%) and recovery (25%) rates were achieved by pre-treating ECs on 0.75 M sucrose for three days followed by dehydration for 20 h, prior to liquid nitrogen immersion. Ultra-structural studies showed no aberrations or damages in the exterior regions of cryopreserved EC. Massive damages at the interior regions after cryopreservation suggested that osmotic and cryo injuries occur in the internal regions of ECs. DNA banding patterns at SSR marker loci showed no evidence of somaclonal variations in tested material confirming that encapsulation-dehydration have not caused any genetic variation in EC.