Genetic stability of mulberry germplasm after cryopreservation by two-step freezing technique

Genetic stability of Morus alba, Morus indica, Morus laevigata (indigenous collection) and Morus species (exotic collection) have been studied in in vitro regenerated plants of mulberry (fresh, before and after cryopreservation) using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers. This study examined the genetic stability of cryopreserved dormant buds of Morusgermplasm that were stored in liquid nitrogen using two-step freezing, then rewarmed and regrown. Dormant buds of mulberry collected during winter period were found suitable for the cryopreservation in liquid nitrogen. In the present study, the plants were regenerated directly from dormant buds (before and after cryopreservation) without intermediary callus phase. These regenerants thus bear low risk of genetic instability. Both the single primer amplification reaction (SPAR) markers showed reproducible and well resolved banding patterns in mulberry germplasm, in which RAPD marker generated a total of 201 bands based on 15 primers; however, ISSR markers were given 145 bands using 11 primers. Both markers showed monomorphic banding patterns and did not reveal any polymorphism among the mother plant and in vitro regenerants before and after cryopreservation, suggesting that cryopreservation, using two-step freezing, does not affect genetic stability of mulberry germplasm. The transitory nature of these polymorphisms should be carefully considered when monitoring for genetic stability.   Key words: Cryopreservation, Genetic stability, in vitro culture, ISSR, mulberry, RAPD.