The story of plastid inheritance in alfalfa began with research with chlorophyll-deficient mutants in the mid 1980s (Smith et al., 1986). Working with two mutants, we showed first that the chlorophyll-deficient phenotypes involved were not likely cause d by a virus since they could not be sap transmitted. Then we demonstrated that the mutants were true breeding and exhibited developmental patterns in sectored tissue characteristic of the sorting-out of normal and abnormal plastids. Lastly, results of re ciprocal crosses using flowers from both pure sectors of both plastid types (Table 1) strongly suggested that the chlorophyll deficiencies were the result of plastid mutations, and that plastids are inherited biparentally with a strong paternal bias.

Following the research of Lee et al. (1988; 1989), a series of reports appeared where analysis of cpDNA restriction fragments was used to describe plastid inheritance in alfalfa. Johnson and Palmer (1989) demonstrated heteroplasmy within population s of alfalfa and Medicago scutellata. They further found a single M. scutellata plant that was heteroplasmic suggesting that it resulted from a mating where both parents contributed plastids. Schumann and Hancock (1989) and Masoud et al. (19 90) observed biparental inheritance of cpDNA and further established a strong pollen parent bias to plastid inheritance in alfalfa.

Establishing a genetic basis for variation in plastid inheritance

We had noted what appeared to be the influences of parental genotype on plastid transmission in our initial work with chlorophyll-deficient mutants (Table 1)(Smith et al., 1986). In order to establish whether this variation was under genetic contro l, I introduced the original yellow-green plastid mutant into four different nuclear backgrounds (Smith, 1989b). Pollen from flowers in pure chlorophyll-deficient sectors on these F₁ plants was then used in crosses with five normal maternal par ents and the plastid constitution of the resulting progenies was scored in seedlings. This research reconfirmed that plastid inheritance in alfalfa is strongly paternally biased and further established that it is influenced by both the maternal and patern al genotypes (Table 2). Particular plants could then be characterized in terms of their average plastid "transmission strength" as a paternal or maternal parent. The high proportion of progenies with apparently only paternally or maternally derived plasti ds observed (mean=68%) suggested that variation in plastid inheritance might be exerted through effects on the number or distribution of plastids from each parent early during embryo development. Detailed ultrastructural analysis was necessary to confirm this.

It is important to note that plastid inheritance depends on transmission and multiplication of cpDNA (plastid genomes) not only of the organelles. It is possible that the apparent lack of a relationship between the number and size of plastids in genera tive cells and transmission strength could be due to differences in the number of plastid genomes contained within each organelle. By examining generative cells stained with the DNA-flourochrome DAPI and using epifluorecence microscopy, it is possible to quantify plastid DNA aggregates ("plastid nucleoids") that presumably represent plastid genomes (Corriveau and Coleman, 1988; 1991). Shi et al. (1991) used this technique and showed that plastid nucleoid number per generative cell was in fact less than th e number of plastids per generative cell suggesting some plastids in this cell may not contain cpDNA. However, as was the case with plastid number, there was no consistent correlation between the number of plastid nucleoids per generative cell and paterna l plastid transmission strength.

By combining the two analysis methods discussed above, Zhu et al. (1992) were able to describe the number and position of plastids and plastid nucleoids in individual sperm cells of alfalfa. Sperm dimorphism could affect organelle inheritance if one sp erm preferentially fertilizes the egg (Mogensen, 1992). However, this research showed that the sum of the number of plastids and plastid nucleoids within the two sperms of a pair is not different from that in the entire generative cell. But it was observe d that over 60% of the plastids in the sperm cells did not contain any DNA based on DAPI staining. Importantly, the number of plastids also did not differ significantly between the two sperms of a pair, confirming that sperm dimorphism does not exist in a lfalfa.

Probably the most revealing observation from all the ultrastructural studies of plastid inheritance in alfalfa came from analysis of egg cells by Zhu et al. (1993). Cells from two genotypes were used in this research. One (6-4) is a strong transmit ter of plastids as a maternal parent while the other (CUF-B) is a weak transmitter. Within the egg cell, plastids in 6-4 were larger and more numerous than in egg cells from CUF-B. Furthermore, more plastids were positioned in the apex of the egg cell in 6-4 than in CUF-B. CUF-B had a majority of its plastids on the micropylar side of the division plane of the zygote. The embryo is derived from only the apical portion of the egg cell in alfalfa, and so these observations would appear to at least partially explain differences between genotypes in maternal plastid transmission strength. Rusche et al. (1995) provided further support for this interpretation by observing that the plastid distribution seen in CUF-B egg cells is maintained through the first divi sion of the zygote. This research also demonstrated that more paternal plastids appeared in the apical portion of the zygote and in the apical cell of the proembryo than did maternal plastids. Collectively, these studies suggest that distribution of mater nal and paternal plastids in the zygote may be a significant determinant of plastid inheritance patterns in alfalfa.

The evolutionary significance of particular modes of plastid inheritance– especially biparental inheritance with a strong paternal bias–remains largely a matter of speculation. Transmission of plastids via the microgametophyte may be related to rol es played by either the organelles themselves or their nucleic acids in affecting reproductive success through their influences on the growth and development of the gametophyte itself. If this is the case, quantitative differences in plastid or cpDNA cont ent might be related to microgametophyte or embryo performance. Further, these differences might then be related to differences in mating system, plant habit, or reproductive success. Keys et al. (1995a) quantified plastid nucleoids in genotypes from gene rative cells in 18 taxa of Medicago and related genera. The number of nucleoids was found to be proportional to volume of pollen grains and generative cells in each genotype studied. When represented relative to these volumes, nucleoid number per g enerative cell was not significantly related to ploidy level, mating system, perenniality (Table 3) or to floral, pollen grain, or generative cell size. These data strongly suggest that the number of plastid nucleoids in the microgametophyte provides no o bvious selective advantage.

Keys et al. (1995b) further investigated the possible relationship between plastid nucleoid number per generative cell and reproductive success in alfalfa. This research showed that the number of plastid nucleoids per generative cell was actually ne gatively associated with male fertility suggesting that sperm that have minimized their plastid DNA content may actually be more successful in affecting fertilization (Fig. 1). We hypothesize that plastid nucleoids may persist in generative cells of a lfalfa in spite of this relationship as a result of the reduced importance of sexual reproduction due to this species perenniality and perhaps its long history of cultivation.

The possibility remains that paternally biased plastid inheritance exists in species such as alfalfa because it provides an opportunity for pollen donors to gain a competitive advantage within a population by affecting progeny performance through impro ved (e.g., more efficient) plastid-nuclear interaction. This represents a potential example of sexual selection (Willson, 1994). Given this species high pollen grain:ovule ratio, (Small, 1988) being able to influence progeny fitness by contributing both p lastids and nuclear genomes allows pollen donors to contribute to the evolution of the population disproportionately to their seed production potential. Such a situation could be envisioned to evolve under a particular set of demographic conditions. Speci fically, breeding populations that are dominated by a relatively few large mature individuals that serve as the primary maternal parents but that may receive pollen from any of a large number of relatively small immature individuals.

While the necessity for a compatible relationship between plastid and nuclear genomes is well accepted (Scott et al., 1991; Glick and Sears, 1994), recognition and exploitation of agronomically positive cytoplasmic-nuclear interactions has not been com mon (Smith, 1989a; Jan, 1992). In our research we have observed significant differences in fitness-related traits such as shoot biomass among reciprocal cross progenies in alfalfa (Table 4). Nevertheless, we have not yet directly linked these differences to interactions between plastid and nuclear genomes. An ongoing experiment may provide data helpful in determining whether the sexual selection scenario outlined above may occur in alfalfa. In this experiment, we have conducted mass selection for forage y ield using equal overall selection intensity but with different intensities imposed upon maternal and paternal parents (Table 5). If paternal parents are able to make contributions to progeny performance that exceed those of maternal parents then progenie s derived from more intense selection of paternal parents should have highest levels of performance. In addition to alfalfa, a parallel selection and evaluation experiment is being conducted with birdsfoot trefoil (Lotus corniculatus), which exhibi ts maternal inheritance of cpDNA (Gauthier et al., 1997).

Evidence regarding the inheritance of mitochondria

The preponderance of data available now indicates that mitochondrial DNA (mtDNA) and most likely mitochondria are inherited in a strictly maternal fashion in alfalfa. Schumann and Hancock (1989) and Forsthoefel et al. (1992) showed that only mtDNA rest riction fragments characteristic of the maternal parent were observed in F₁ hybrids in alfalfa. Mitochondria have been observed in generative and sperm cells of alfalfa (Zhu et al., 1991). However, Zhu et al. (1992) reported the number of these organelles was greatly reduced during sperm cell development and suggested this may be the result of mitochondrial degeneration. Observing distinctly different modes of inheritance for these two primary organelles is not at all unusual in plants. Indeed, it is generally the rule (e.g., Mogensen, 1996; Testolin and Cipriani, 1997; Havey et al., 1998), and the complicates evolutionary explanation of modes of organelle inheritance.

The authors would like to recognize and thank those who have exerted significant influences on the authors of this article and on much of the research it describes. At the University of Wisconsin: E. T. Bingham, R. W. Groose, W. P. Kojis, and L. E. Talbert; at Montana State University: N. Blake, T. K. Blake, D. J. Lee and T. J. McCoy; at Northern Arizona University: M. Rusche, L. Shi and T. Zhu; at the University of Arizona: H. J. Bohnert, D. J. Fairbanks, D. Fendenheim, N. R. Forsthoefel, and R. N . Keys; at Michigan State University: B. B. Sears; and at the University of Wales: R. E. A. Tilney-Bassett.

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Fig. 1. Path analysis diagram describing microgametophytic characters and components of male reproductive success for eight genotypes of alfalfa when crossed with the male-sterile genotype 6-4. Double-he aded arrows depict correlations and numerical values are path coefficients. Single-headed arrows depict cause-and-effect relationships and numerical values are standardized regression coefficients. (Adapted from Keys et al., 1995b).

Table 1. Number of normal and chlorophyll-deficient progenies produced in reciprocal crosses between normal testers and pure yellow-green and albino flowers on mutant plants. (Adapted from Smith et al., 1986).

Table 2. Percentage of purely paternal, purely maternal and sectored progenies from crosses between two chlorophyll-deficient (CD) and two normal (green, G) alfalfa plants. (Adapted from Smith, 1989b).