Red clover (Trifolium pratense L.) is an important forage legume grown in many of the temperate regions around the world. In order to evaluate possible causes for the low seed yield in particular in tetraploid red clover, we surveyed 71 studies of red clover seed production published from 1948 to 2019, reviewing seed yield components, pollination and reproductive traits. Seed yield components are recorded to contribute to and improve our understanding of the complex processes leading to the red clover seed yield. However, the measurement approaches varied largely among the studies, depending on whether the seed yield components are directly measured or calculated. Red clover seed production is dependent on insect pollen transfer. However, much uncertainty of pollination and reproductive success remains. Pollination conditions were often neglected: one third of the papers did not mention pollination conditions. The information that connects pollination and seed set are lacking, very few studies measured traits in floral morphology, ovule fertilisation and seed maturation. We suggest that consistent and comprehensive evaluation of pollination and reproductive success are required to improve our understanding of reasons for the low seed yield in red clover and hence the possibilities for future improvement.

Plant reproduction in red clover requires cross-fertilization via insect pollination. However, the influences of visitation rate and timing on maximizing ovule utilization are yet to be determined. We aimed to study the influences of visitation rate, flowering stage, and self-incompatibility on reproductive success. We applied hand and honey bee pollination in the study of eight red clover cultivars with two ploidy levels released between 1964 and 2001. In hand pollination, increasing the visitation rates (from 10 to 80 pollinated florets per flower head) increased the seed number per flower head but reduced the seed number per pollinated floret. Different flowering stages (early, middle, and full flowering) did not influence the seed number per pollinated floret significantly. There was a marked difference in reproductive success depending on the ploidy level, with 0.52 seeds per pollinated floret in diploid and 0.16 in tetraploid cultivars. During the cultivar release history, seed number per pollinated floret seemed to decrease in diploid cultivars, whereas it increased in tetraploids. In honey bee pollination, diploid cultivars had more two-seeded florets than tetraploids. Different visitation rates and the stochastic nature of pollen transfer resulted in difficulties when the plant reproductive success between hand and bee pollination was compared. A maximum of 0.27 seeds per pollinated floret were produced in hand pollination compared to the 0.34 in honey bee pollination. In spite of this, hand pollination provided a valuable method for studying the pollination biology and reproduction of red clover. Future studies may employ hand pollination to unravel further aspects of the low reproductive success with the future perspective of improving seed number per pollinated floret in tetraploid red clover.

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 of honey bees (one bee, two bees and open pollination) and pollen diversity (one and two pollen donors) of 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 ‘Rajah' (on average 20). Seed set in ‘Amos' was lower than 50% of ‘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.

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High and stable seed yield is critical for red clover (Trifolium pratense L.) seed production and the commercial exploitation of the crop. A three-year experiment was conducted from 2013 to 2015 under Danish field conditions to explore the influence of precipitation during peak flowering on the seed yield of three red clover cultivars. We investigated the flowering duration and intensity based on a visual scale assessment, seed yield, and thousand seed weight in all three experimental years. In 2014 and 2015 we measured the seed yield components of floret number per flower head, seed number per flower head, and seed set. During the experimental period, high seed yields of more than 1000 kg ha−1 were obtained for the diploid cultivars ‘Rajah’ and ‘Suez’. Although a relatively high seed yield of 500 kg ha−1 was obtained in the tetraploid cultivar ‘Amos’, this was only around half of the seed yield and seed set of the diploid cultivars. Precipitation during peak flowering positively influenced the seed yield and thousand seed weight for the three cultivars. We conclude that observations of flowering phenology are required to determine the impact of environmental conditions on seed yield in red clover cultivars. Further, adequate water supply during peak flowering is important to obtain the high seed yield of red clover.