A two-step freezing cryoprotocol preceded by desiccation to 15 to 25% moisture content was developed and successfully applied to winter dormant buds of mulberry (different Morus spp.) of a core set comprising 238 accessions studies in our laboratory. The survival and recovery percentage of diverse accessions cryobanked for various periods were tested under in vitro conditions, and several factors were analyzed to determine their role in optimizing the recovery of low-viability accessions. The effect of rates of freezing and thawing (both fast and slow), were tested and recovery compared. Recovery conditions such as dark incubation and rehydration in sterile moist moss grass for different durations after cryopreservation led to a higher survival percentage compared to controls. Two different recovery culture media were compared for their efficiency in survival. On average, the survival under in vitro culture conditions using optimized conditions was high: above 60% in majority of the accessions. Dormant buds showed viability in the range of 25 to 100% with an average of 50.4%. The recovery percentage of winter dormant buds after cryopreservation via slow freezing and slow thawing with rehydration by moist moss grass for 2 h was recorded in the range from 63.3 to 90.9% with an average of 81.05%. Without rehydration, it ranged from 50 to 75% with an average of 60.4%. Regeneration of cryopreserved mulberry germplasm after 6 years of storage indicated no survival loss over different years of storage, and 33-40% of the accessions showed viability above 40%, up to a maximum of 100%. Maximum shoot formation (100%) was obtained from Morus alba. The majority of the accessions were rooted in vitro within 20-25 days of subculture in the auxin rich rooting media, except in wild species M. latifolia and M. laevigata, which took longer (45 to 60 days) for root development. All the rooted plantlets were then transferred to the field and successfully established in a glasshouse.
Keywords: Morus spp.; cryopreservation; dormant buds; in vitro regeneration; recovery; two-step freezing.