Red clover is an important fodder crop. Therefore, it is included in most grassland mixtures for mowing. It is popular because it is very productive and can better withstand drought than e.g., white clover.

“There are both diploid and tetraploid varieties, where the production of dry matter is highest in the tetraploid varieties, but unfortunately the seed yield is 20-50% lower than in the diploid types. And with a seed yield in red clover, which has been declining compared to white clover since the mid-90s, it is critical for the distribution of the tetraploid varieties that the seed yield is so much lower than in the diploid varieties,” says senior researcher Birte Boelt from the Department for Agroecology at Aarhus University.

Together with former PhD student Shuxuan Jing, she has investigated whether pollination conditions play a role in seed yield. It can e.g., be how many times each flower is visited by pollinating insects, and how far the flower is in its stage of development when it is pollinated.

Diploid and tetraploid varieties

There are many different varieties of red clover. The original form is diploid, but there are also refined tetraploid varieties. The latter has been in production since the 1940s. Using plant breeding techniques, the chromosome set in the plant was doubled with the aim of improving e.g., harvest yield and disease resistance. The tetraploid varieties also have a higher yield when it comes to feed, but for the seed yield this is unfortunately not the case. Seeding in tetraploid varieties is between 20 and 50% lower than in diploid varieties.

"We want as many species as possible for seed breeding in Denmark, but the challenge is that we do not know why the seed yield in red clover decreases and why it is so much lower in tetraploid varieties," says Birte Boelt.

Pollination with hands and bees

Often tetraploid varieties have a larger flower, but the number of small flowers is the same as in the diploid varieties. A red clover flower typically consists of 90-110 small flowers per flower head, and in each small flower there are two aptitudes for seeds. That is, there is potential for up to 220 seeds per red clover flower.

"However, it is far from every small flower that sets seeds and only very few of them utilize the potential to form two seeds," explains Birte Boelt.

In a field experiment, the number of flower seeds per flower head was counted, and here a very large variation was found from 20 to 140 seeds per flower head. In seed breeding, one depends on how many bees are present in the individual field, just as the weather during flowering also plays a big role. There is a widespread suspicion that the actual pollination may play a role in relation to the large difference in seeding. Therefore, the researchers have also worked with different types of pollination:

• Hand pollination
• Pollination with honey bees
• Pollination with bumble bees

Hand pollination takes place by manually moving fresh pollen from one flower head to another with approx. the same number of pollens for each anther. After pollination, the flower head is covered with a small bag, and when the flower has matured, it is harvested and dried, after which the number of seeds is counted.

Pollination with honey bees is measured in a tunnel with red clover in pots and a small herd of honey bees. In the experiment, the diploid and tetraploid plants were visited just as often by the bees. After pollination, the flower heads were also covered with small bags and examined in the same way as in the experiment with hand pollination.

In the fields at Aarhus University's research center in Flakkebjerg, red clover, white clover and alfalfa have been grown for seeds in the same area. The area is frequently visited by various species of bumble bees. The garden bumblebee preferred the tetraploid red clover, but the other bumble bees visited both varieties equally diligently.

Still lower seeding

One of the aims of the experiments was to investigate whether more frequent visits from pollinators would be able to increase the number of seeds. In the experiment with hand pollination, the researchers found that with an increasing visitor rate of up to 80 small flowers per flower head, the total number of seeds in the flower head increased, but not per small flower.

"We also found that it does not matter in relation to seeding how far in the flowering stage the red clover flower is when it is pollinated," explains Birte Boelt.

Even under optimal and exactly the same pollination conditions, it was found that diploid red clover sets up to three times as many seeds per pollinated small flower as tetraploid varieties.

“We cannot rule out that pollination may be the cause of the difference between the two types, but it seems more likely that it is due to genetic differences. It is therefore up to the plantbreeders to find a solution to the problem,” says Birte Boelt, who together with Shuxuan Jing last year presented the results to nine European red clover breeders and representatives from seed breeding.