Background: Asynchronous flowering is one of the major constraints for hybridization between Abelmoschus moschatus subsp. moschatus, a wild species closely related to cultivated okra [A. esculentus (L.) Moench]. Availability of viable pollen is a prerequisite to facilitate breeding in these species.
Objectives: Pollen cryopreservation was attempted in A. moschatus subsp. moschatus, to overcome the asynchronous flowering barrier during wide hybridization with A. esculentus.
Materials and methods: Viability of fresh pollen of A. moschatus subsp. moschatus was assessed using acetocarmine and triphenyl tetrazolium chloride (TTC) test and in vitro germination by sitting drop culture method. Pollen of 10 accessions were stored at four temperatures (25, 4, -20 and -196 degree C), in the dark and periodically monitored for viability. The standardized cryopreservation protocol was applied to 24 accessions of A. moschatus subsp. moschatus over three months. In vivo pollen germination of 24 accessions of cryopreserved pollen and its efficacy on fertilizing A. esculentus cv 'Pusa Sawani' were recorded and pollen was utilized for hybridization with A. esculentus.
Results: Brewbaker and Kwack medium with 15% sucrose was optimal for in vitro pollen germination. Pollen viability assessed by in vitro germination (60-90 %) was more reliable compared to acetocarmine (90-99 %) and TTC (85-99 %) staining tests. Significant negative correlation was found between pollen germination, storage time and temperature (25, 4 and -20 degree C) in all the accessions. Cryopreserved (-196 degree C) pollen showed significantly higher viability compared to all the other storage conditions, without viability loss. Successful pollination, fruit and seed set was observed in four out of 24 cross combinations attempted.
Conclusion: The cryopreservation of pollen of A. moschatus subsp. moschatus and its fertilizing ability offers great potential for a successful wide hybridization programme in okra.