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16-chromosome forms of red clover (2n=14+2) were crossed to six Trifolium species with the chromosome number 2n=16. Hybrid plants were derived from the cross of a stable 16-chromosome red clover T. pratense with T. diffusum (2n=16). No seeds were obtained from reciprocal crosses. F₁ hybrid plants were morphologically more similar to T. diffusum, whereas their other characters, e.g. flower number per head, were intermediate between the species crossed. All F₁ hybrids had the chromosome number 2n=16. Meiosis proceeded with large irregularities. The average number of bivalents per cell was 3.32, and that of univalents - 9.98. Univalents exhibited a high stickiness and frequently formed "end-to-end" configurations and chains consisting of about a dozen of so chromosomes. Bivalents were straight. Lagging chromosomes and chromosome bridges were observed during AI; lagging chromosomes were also found during AII. After an equalizing division, tetrads and different from them microspore polyads were formed. 16-chromosome hybrids were male- and female-sterile. No hybrids were obtained from the stable 16-chromosome red clover T. pratense (n=7 chromosomes) crossed to the selected clover species (T. apertum Bobr., T. alexandrinum L. and others) with n=8 chromosomes.
After duplicating the chromosome number by colchicine in sterile F₁ 16-chromosome hybrid T. pratense × T. diffusum some partially fertile plants with 32 chromosomes were found. Male fertility (viability of pollen grain) was from 69.3% to 86.2% (on average 81.8%), whereas female fertility estimated as seed setting after cross- and self-pollination was 21.8% and 6.9%, respectively. Male and female fertility as well as somatic chromosome number were examined in the F₂-F₄ generations. Selection for female fertility resulted in increasing seed setting in the first two generations (F₂ and F₃) and in decreasing it in F₄ generation. An average seed setting in the F₂-F₄ generations after cross-pollination amounted to 22.2%, 43.6% and 12.9%, respectively; after self-pollination it was 25.2%, 27.6% and 1.9%. In the F₂ generation all the plants had 2n=32 chromosomes. In the next generations there appeared aneuploids, among which 30-chromosome individuals were predominant.
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