Greater complementary gene interaction in autotetraploid alfalfa (Medicago sativa L., 2n = 4x = 32) may explain differences in vigor and breeding behavior between diploids and autotetraploids. Complementary gene interaction is nonallelic gene interaction or epistasis where dominant alleles at heterozygous loci may complement each other by masking recessive alleles at respective loci. This paper describes how tetrasomic segregations of linkage blocks in linkage disequilibrium produce tetraploid individuals and populations with greater complementary gene interaction than is possible at the diploid level. This finding helps explain autotetraploid superiority and unique breeding behavior. Research on gene action in autotetraploid alfalfa has demonstrated that favorable alleles in linkage blocks underpin population improvement and increased heterosis. The individual favorable alleles with additive effects also contribute to non‐additive complementary gene interactions in linkage blocks. Apparent multiple allelic interaction (overdominance) effects discussed in earlier studies inbreeding depression and progressive heterosis in alfalfa are due mainly to linkage disequilibrium, which agrees with findings in maize. The severe inbreeding depression in autotetraploids is due mainly to the rapid loss of complementary gene interactions in the first few generations of inbreeding. Correspondingly, the progressive heterosis of autotetraploids is due mainly to a progressive increase in complementary gene interactions. Greater complementary gene interactions in tetraploid alfalfa also helps explain recent DNA research indicating that yield in tetraploids is more responsive to genetic diversity than in diploids. Many differences between diploid and autotetraploid alfalfa reported in earlier studies now may be explained by inherent differences in the levels of complementary gene interactions.

Polyploidy appears dependent on heterozygosity! The largest group of po1yp1oids, the a11opo1yp1oids (disomic po1yp1oids), have fixed heterozygosity in the two or more divergent genomes they possess (e.g., wheat, oats, cotton, tobacco, etc.). Hexaploid wheat, for example, although self-pollinated and basically homozygous at loci in each of its three genomes, has internal hybridity among loci with similar function in its three genomes. Disomic po1yp1oids thus are able to capitalize on the merits of both the se1f-and cross-fertilizing systems (1).The autopo1yp1oids (polysomic po1yp1oids) insure their heterozygosity through cross-pollination (e.g., alfalfa, birdsfoot trefoil, potato, and many grasses). We can find no example in crop plants of a successful polysomic polyploid species which is self-pollinated. Evidently the polysomic condition can not tolerate the homozygosity associated with self-pollination. The biochemical and physiological advantages of heterozygosity must be an important component of polyploid vigor (2). Polysomic po1yp1oids are represented by segregating and heterogeneous populations where maximum heterosis may be expressed by a few elite individuals in the population but not by the population per se. (Apomixis can preserve and perpetuate the elite polyploid individuals but will not be reviewed here.) In our seed reproduced polysomic po1yp1oids we can maximize the frequency of elite genotypes, but at present we cannot fix such genotypes in cultivated populations. As will be illustrated in this paper, maximum heterozygosity and heterosis does not occur in the Fl or single cross generation when parents are inbred (as it does 1n diploids or disomics) but occurs in the segregating double cross or an even later generation. Under polysomic polyploid conditions, the more inbred the parents, the lower the 471 W. H. Lewis (ed.), Polyploidy

Tetraplold alfalfa (Medicago sativa L.) germplasm, in which two‐thirds of the plants are capable of regeneration from callus tissue in culture, has been developed by recurrent selection. It descends from one ‘DuPuits’ and four ‘Saranac’ clones which regenerated in the cycle 0 population. Frequency of regeneration was 12% in cycle 0, about 50% in cycle 1, and 67% in cycle 2. Seed produced by hand‐intercrossing 75 plants regenerated from cycle 2 has been termed ‘Regen‐S’ (Regen for regeneration; S for Saranac). It is purple‐flowered and phenotypically similar to Saranac. One‐gram lots of Regen‐S seed are presently available for distribution, and larger amounts are expected to be available in late 1975. A yellow‐flowered regenerator ‘Regen‐Y,’ is now being bred from selections out of ‘Rhizoma,’ ‘Rambler,’ and ‘Drylander.’ Small amounts of Regen‐Y seed are also presently available, thus providing a contrasting qualitative genetic marker stock.

Four diploid (2x) clones of alfalfa, Medicago sativa L., which produced good seed set when used as male parents in 4x-2x crosses were selected for study. The 2x clones descended from 2x haploids of cultivated 4x alfalfa. Fertility in the 4x-2x cross was due to the production of pollen with the unreduced chromosome number (2n pollen) from the 2x parent. The cytological mechanism of 2n pollen formation was found to be disorientation of spindles at metaphase II in up to 38% of the pollen mother cells. Thus, both n and 2n pollen were produced by all four diploids examined. Normal spindles at metaphase II were oriented such that they defined the poles of a tetrahedron and resulted in normal tetrads in a tetrahedral arrangement. Disoriented spindles were basically parallel to each other and resulted in formation of dyads and occasionally a triad. Dyads developed into two 2n pollen grains; triads developed into one 2n and two n pollen grains. Since both n and 2n pollen grains are produced by the diploids, they can be maintained as diploids or they can be used as male parents in crosses to tetraploids. The genetic constitution of 2n pollen resulting from parallel spindles is similar to that expected after first division restitution of meiosis and much of the heterozygosity of the diploid parent is conserved in the gametes. The 2n gamete mechanism has potential application in germplasm transfer and in maximizing heterozygosity in tetraploid hybrids.

Changing climates and associated increased variability pose risks to alfalfa (Medicago sativa L.) cultivation, with the requirement to establish, survive, and maintain production under water stress. Crop wild relatives (CWR) of alfalfa include populations that have evolved to survive in a number of different, extreme environments, but until recently have had limited use in breeding programs. Here we report on the phenotypic diversity of alfalfa crop wild relatives that were selected to represent extremes in drought tolerance (by sourcing germplasm from environments with extremes in low rainfall, high temperature, shallow soils, and winter freezing) with the aim of providing germplasm with drought tolerance and improved forage yield traits for breeding programs in both warm and cool dry temperate environments. Newly formed hybrids created between M. sativa, M. arborea L. (a woody shrub), and M. truncatula Gaertn. (an annual species from the Mediterranean region) were developed or acquired to introduce new genetic diversity from the tertiary genepool. Preliminary characterization and evaluation was used for taxonomic classification, and to identify wild accessions and pre‐bred (hybrid) lines that offer new diversity for growth habit, seed size, fall dormancy, and forage yield. The accessions and pre‐breeding lines described have been donated to the Australian Pastures Genebank for conservation and distribution.

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The cytological mechanism of 2n egg formation was studied in several diploid (2n = 2x = 16) and tetraploid (2n = 4x = 32) clones of cultivated alfalfa (Medicago sativa L.). The comparison of normal megasporogenesis with megasporogenesis that produced 2n eggs was made using an ovule clearing technique with methyl salicylate. Developmental sequences in the formation of n and 2n eggs were the same through anaphase II. Following anaphase II in 2n egg formation cytokinesis occurred only in the micropylar diad, not in the chalazal diad. Micropylar megaspores disintegrated leaving a functional unreduced megaspore of the second division restitution (SDR) type at the chalazal end. The two nuclei in the megaspore can fuse prior to the mitotic divisions or during the first two mitotic divisions. The SDR mechanism of 2n egg formation was confirmed in selected diploid clones by comparing half-tetrad analysis of 2n eggs with half-tetrad analysis of a known first division restitution (FDR) 2n pollen producer. During the study of 2n egg formation in tetraploid alfalfa, several diploid clones were found which produced octoploid (2n = 8x = 64) progeny from 4x-2x crosses. Pollen development in the diploids was normal through telophase II. Cytokinesis was absent, however, and the four telophase nuclei fused to produce a 4n pollen grain. The fusion of a 2n (2n = 4x = 32) egg and a 4n (4n = 4x = 32) pollen grain produced the octoploid progeny.

Transgenic alfalfa plants expressing Bacillus licheniformis alpha-amylase and manganese-dependent lignin peroxidase (Mn-P) from Phanerochaete chrysosporium were produced using the Agrobacterium tumefaciens transformation system . In each case, there was a range of expression of the introduced gene among independent transgenic plants . Plants producing alpha-amylase showed no alteration of phenotype . Production of Mn-P in alfalfa, however, in most cases adversely affected plant growth and development . Affected plants were stunted with yellowing foliage, but survived and produced seed . Results from field trials showed that Mn-P production in transgenic alfalfa reduced dry matter yield and plant height . The extent of these symptoms and yield reduction was, for the most part, related to the level of foreign protein production as estimated by Western analysis . Field data from transgenic plants expressing alpha-amylase showed that there was no effect of foreign protein production on plant performance . Expression of Mn-P was shown to segregate in sexual progeny derived from transgenic plants .Abbreviations : Mn-P -manganese-dependent lignin peroxidase