Production of 2n Pollen in Red Clover1

The success of tetraploid red clover (Trifolium pratense L.) in Europe has stimulated interest in developing tetraploid red clover adapted to the United States. The use of 2n gametes to produce tetraploid alfalfa (Medicago sativa L.) and potato (Solanum tuberosum L.) from diploid germplasm suggests that 2n gametes may be an alternative to chemical treatment. Diploid plants that produce 2n eggs are known to occur in red clover, and plants that produce 2n pollen have been reported. The objectives of this study were to search for red clover plants producing 2n pollen, and to determine the cytological origin of such pollen. Six hundred plants, belonging to six different diploid (2n = 2x = 14) cultivars, were screened for 2n pollen production by examining dry pollen samples with the aid of a microscope. Eighteen plants (3%) produced at least 1% 2n pollen and were classifted as 2n pollen producers. Individual plants ranged from 1 to 84% in frequency of 2n pollen production. Pollen mother cell analyses revealed that 2n pollen resulted from parallel or tripolar spindles during anaphase II of microsporogenesis. This mechanism is genetically equivalent to a first division restitution (FDR) mode of gamete formation. One 2n pollen producer proved to be a synaptic mutant. Normally, synaptic mutants are male sterile, but fertility is restored by parallel spindles in this plant. Because the lack of homologous pairing limits genetic recombination, this plant can produce gametes with the same genetic constitution as the sporophyte. It is apparent from the results that 2n gametes may be useful in red clover polyploidization programs. Additional index words: Parallel spindles, Trifolium pratense L. , Synaptic mutant, Tetraploid red clover, 2n gametes. R clover (Trifolium pratense L.) is a diploid (2n = 2x -14) species used as a forage crop . throughout the temperate regions of the world. There Is no evidence to date of naturally occurring tetraploid forms of the species; however, chemically induced tetraploids (2n = 4x = 28) have been produced. In certain parts of Europe, tetraploid forms are sometimes agronomicall~, superior to the diploid forms in yield, disease resistance, and persistence (Taylor and Smith, 1979). Considering the success tetraploids in Europe, it may be desireable to develop . tetraploid germplasm adapted to United States growlng conditions. The conventional method for obtaining red clover tetraploids has been through the use of colchicine or nitrous oxide. Recent research with 2n gametes in other species suggests that these gametes can be used as an alternative to chemical treatments in a breeding program. Production of 2n pollen has been extensively studied in potato, Solanum tuberosum L. , (Mok and Peloquin, 1975), alfalfa, Medicago sativa L. , (Vorsa and Bingham, 1979), and peas, Pisum sativum L. , (Myers, 1981). First division restitution (FDR) 2n len has facilitated the transfer of germplasm from the diploid level to the tetraploid level in both potato 1 Cooperative investigation of USDA-ARS and the Wisconsin Agric. Exp. Stn. , Madison, Wis. This research supported in part by U.S. Competitive Grant no. 8100349. Received 11 |uly 1983. ~ Research assistant, Dep. of Agronomy, Univ. of ~Visconsin, Madison, and supervisory research geneticist, USDA-ARS and professor of agronomy, Univ. of Wisconsin, Madison, WI 53706. (Peloquin, 1982) and alfalfa (Bingham, 1968). In these species, 2n pollen can be produced by disorientation of the meiotic spindles during anaphase II. The spindles may lie in one plane instead of two, in which case they are either parallel or tripolar. Parallel s.pindies lead to the formation of two restitution microspores, while tripolar spindles form one restitution microspore and two normally . reduced microspores. The]~roduction of 2n pollen via parallel or t~:~polar spindles is genetically, equivalent to the FDR mechanism of 2n gamete tormation. All the heterozygous loci from the centromere to the first crossover are preserved in each chromosome arm, 50% from the first crossover to the second, and 75% in each successive crossover. Thus, most of the heterozygosity present in the sporophyte is retained in the gametophyte and subsequently transmitted to the progeny (Mendiburu and Peloquin, 1977). A synaptic mutant, occurring together with parallel spindles during anaphase II, has been described in potato. The combination of these two mutants leads to the formation of pollen with exactly the same genetic constitution and heterozygosity as the sporophyte. This occurs as the lack of normal pairing significantly reduces crossing over, resulting in a corresponding increase in the amount of heterozygosity transmitted by a restitution gamete. When the lack of pairing is complete the genome of the gametophyte is identical to that of the sporophyte (Peloquin, 1983). Maximizing the heterozygosity in autopolyploids is important (Dunbier and Bingham, 1975), and FDR 2n gametes can be an effective breeding tool for autopolyploid crops (Mendiburu et al., 1974; Bingham, 1979). Red clover plants that produce 2n eggs have been identified (Broda and Smith, 1980; Taylor and Giri, 1982), suggesting that 2n gametes can be used to produce tetraploid red clover germplasm. The only report of functional 2n pollen within the true clovers (Trifolium spp.) has been in T. alpestre L. (Maizonnier, 1972). Strzyzewska (1974, 1976) reported the formation of 2n pollen by post meiotic fusion in diploid red clover, but pollen function was not tested in 4x2x crosses. Kazimierski and Kazimierska (1970) reported that a hybrid of T. repens L. X T. nigrescens Viv. produced dyads and triads. The authors also included a microphotograph of tripolar spindles. The objectives of this investigation were to search for plants which produced 2n pollen in diploid red clover and to determine cytologically the origin of the 2n pollen. MATERIALS AND METHODS In red clover, x and 2x pollen can be easily distinguished by their distinct morphology when dry. Haploid pollen is oblong and regular in shape, while diploid pollen is triangular, square, or irregular in shape (Taylor et al., 1976). Red clover flowers were tripped on a slide, and the pollen was then examined under low magnification. One thousand grains from each plant were scored for ploidy level. In all, 600 plants, 100 each from six diploid red clover cultivars,