Isolation of Eggs and Synergids in Ceiba speciosa

Viable egg and synergid cells of Ceiba speciosa were isolated using enzymatic digestion and mechanical dissection. The ovules were digested by 1% (w/v) cellulase RS, 0.3%similar to 0.8% (w/v) pectolyse Y-23, and 0.8% (w/v) hemicellulase for 40-50 min and then transferred for dissection to an isolating solution containing 9% mannitol without enzymes. The two integuments of the ovules were peeled off and the nucellus was cut transversely through the middle. The three cells comprising the egg apparatus (the egg cell and two synergids) were released from the excised nucellus by pushing the micropylar end with a dissecting needle. Dissection of 50 ovules in the isolating solution yielded 19 groupings of egg apparatus cells within an hour. Following this protocol, egg and synergid cells of C. speciosa can be effectively isolated.