Different pollination approaches to compare the seed set of diploid and tetraploid red clover (Trifolium pratense L.)

In red clover seed production, low seed yield is limiting the commercial
exploitation of tetraploid red clover. To explore if pollination is the
limiting factor for the seed yield in tetraploid red clover, we
investigated pollinator behaviour and plant reproductive success in
diploid (2x) cultivar ‘Rajah’ and tetraploid (4x) cultivar ‘Amos’ using
honey bee and hand pollination approaches. We measured the seed set at the
flower head level with the increasing visitation rate in honey bees (one
bee, two bees and open pollination) and pollen diversity (one and two
pollen donors) in hand pollination. We found no difference in honey bee
pollination behaviour (visitation rate) between the diploid and tetraploid
red clover. Surprisingly, the seed set in two bees was significantly lower
than that of one bee in ‘Rajah’. We suggest that honey bees prefer to
visit already tripped florets or to visit fewer florets due to scent marks
left by the previous honey bees. In open pollination, seed number
per flower head in ‘Amos’ (on average 11) was half of the ‘Rajah’ (on
average 20). Seed set in ‘Amos’ was lower than 50% of the ‘Rajah’ in honey
bee (one bee treatment) and hand pollination (one and two pollen donors
treatments). There were no differences in stigma receptivity whereas
pollen viability in ‘Amos’ was lower than ‘Rajah’ after the peak pollen
viability was reached (six days after the onset of flowering). We suggest
that the fertility problems such as low pollen viability may be the reason
for the low seed set in tetraploid red clover. This conclusion should be
verified by investigating other fertility traits during the ovary, embryo
and seed development with a broader range of diploid and tetraploid
cultivars.