Increasing pollination possibilities in Paspalum species: in vitro and in vivo viability of cryopreserved pollen to address flowering asynchrony.

Paspalum is a vital forage and turf grass in tropical and subtropical regions, yet its breeding programs face challenges due to the lack of natural flowering synchronization between some parent species. Pollen cryopreservation offers a potential solution to this issue. This study aimed to adapt a cryopreservation protocol for the pollen of P. atratum, P. malacophyllum, and P. regnellii, and to evaluate the viability of cryopreserved pollen grains (CPG) for hybridization purposes. Two dehydrating agents (LiCl and silica gel) were tested for different durations (30, 60, and 120 min) alongside a non-dehydration treatment. The effectiveness of cryopreservation was assessed over multiple time points (1, 10, 30, 90, 180, 270, and 365 days) with freshly harvested grains as controls. Pollen viability was determined using 0.25% 2,3,5-triphenyltetrazolium chloride staining. Viability of CPG ranged from 40.67 to 80.67% across treatments. Optimal dehydration involved LiCl for 30 min and silica gel for 120 min, achieving an average viability of 66% after 12 months, comparable to fresh pollen. In vivo germination tests confirmed successful pollen tube germination with the combinations P. urvillei × P. malacophyllum; P. urvillei × P. regnellii and hybrid (P. plicatulum 4PT × P. guenoarum cv. Azulão) × P. atratum, although pollen tubes did not reach the micropyle in some crosses. This study established effective pollen cryopreservation protocols for P. atratum and P. malacophyllum, facilitating in vivo germination and enhancing the potential for hybridization in Paspalum breeding programs, thereby addressing flowering asynchrony and broadening crossing opportunities within the genus.