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Biparental inheritance of plastids has been demonstrated in approximately one-third of angiosperm species examined, although the species studied may not be representative of angiosperms in general. Biparental inheritance of mitochondria in angiosperms has been observed in only one case. Since essential cellular functions, as well as several important agronomic traits, are encoded by plant extranuclear genomes, biparental inheritance of organelles has important implications for plant genetics and breeding. Evidence of biparental inheritance of plastids in alfalfa (Medicago sativa L.) suggests the possibility that mitochondria may likewise be inherited biparentally. The objectives of this dissertation were to determine: (1) the inheritance of mitochondria in alfalfa (uniparental or biparental), and (2) the inheritance of cytoplasmic male sterility and the extent of male fertility restoration in single and population crosses of alfalfa. Several large RNA molecules observed in alfalfa mitochondrial preparations were inherited biparentally. These molecules were unaffected by RNase A added to preparations of intact mitochondria indicating that the RNA's were contained within an RNase impermeable compartment. Linear sucrose gradient purification failed to separate the RNA's from mitochondria and examination of sucrose gradient fractions using transmission electron microscopy (performed by J. K. Brown, Dept. of Plant Pathology, Univ. of Arizona) revealed that mitochondrial preparations were free of contamination by virus-like particles and other organelles. These results indicated that the large RNA's were contained within the mitochondrion. The inheritance of large mitochondrial RNA's in alfalfa provided evidence that mitochondria are inherited biparentally in this species. Association of cytoplasmic male sterility with a particular organelle has not been determined in alfalfa, although the mitochondrion has been implicated in several other species. Analysis of progeny from single and population crosses provided evidence of biparental inheritance of cytoplasmic male sterility. Biparental cytoplasmic inheritance as well as nuclear inheritance may influence male fertility restoration.

Inheritance of the multifoliolate leaf (MFL) characteristic (leaves containing more than three leaflets) in tetraploid alfalfa was studied. Also, the effects of temperature and short-term aging of a few weeks on the expression of the MFL trait were determined. Broad sense heritability of the trait was also estimated. Two sets of diallel reciprocal crosses were intially made in the greenhouse using vacuum pump and alcohol for emasculation of the seed parent. There were six different clones in each set. Set I contained ML₁, ML₂ ML₄₉, ML(G), Lew-1, and Hayden-1 clones. The average number of leaflets per leaf for the above mentioned clones was 5.65, 4.52, 3.55, 3.20, 3.00, and 3.00, respectively. Set II was composed of ML₇, ML(F), ML₁₂, ML₅₂, N₁, and N₂ clones with the average number of 4.75, 4.15, 3.50, 3.25, 3.00 and 3.00 leaflets per leaf. Based on the F₁ and S₁ progenies, it was hypothesized that at least three major and independent genes, each with equal contribution to the expression of the MFL trait, were involved. This hypothesis was later substantiated by the observed segregation ratios of the 75 F₂ families from the cross Lew-1 x ML₁ which closely fit the expected ratios. These three genes, LA, LB, and LM showed incomplete dominance with additive gene action. No evidence for epistasis was found. There was a positive association between the number of dominant alleles and the degree of expression of the MFL trait. This association was so specific that plants with less than four, four, five, six, seven or more dominant alleles had an average number of 3.0, 3.1, 3.2-3.4, 3.5-4.0, and 4.1 or more leaflets per leaf respectively. Analysis of the 11 reciprocal crosses between trifoliolate (TFL) and MFL clones showed that only two reciprocal crosses produced a significantly higher number of MFL than TFL plants when the female parent was a MFL clone. Maternal or cytoplasmic effects were negligible. The average frequency of MFL leaves of the two clones (ML(G), ML₂) studied did not significantly change under warm (34°C, 25°C) and cool (17°C, 15°C) temperatures. There was no significant change in the expression of the MFL leaf frequency trait in these two clones from the first harvest scoring to the next. Plants were scored before harvesting at full bloom. Calculated broad sense heritability of the MFL trait was 86%. This indicates that the recurrent selection method for several cycles should increase the frequency of MFL plants in an alfalfa population very rapidly. There are excellent opportunities for improving the quality of commercially grown alfalfa by incorporating the MFL trait.

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Typescript (photocopy). / Digitized by Kansas Correctional Industries

Gene expression in two different genotypes of alfalfa, salt-tolerant and salt-sensitive, was examined by studying differences in protein products coded for by poly(A+) RNA isolated from shoot and root tissue. Plants were grown in hydroponics under unstressed or salt-stressed conditions. Two salinity levels (low salt: 30 mM NaCl and 6 mM CaCl2 and high salt: 133 mM NaCl and 27 mM CaCl2) and one unstressed control were applied. The salt-tolerant genotype showed higher biomass accumulation than the salt-sensitive genotype under both control and salt-stressed conditions. The difference in biomass accumulation between the two genotypes was greatest at the highest salt level. The effect of salt stress on gene expression was studied via in vitro translation of poly (A+) RNA with (35S) -methionine. The labeling pattern was similar in all treatments when analyzed by one dimensional SDS-PAGE. However, a two dimensional analysis (isoelectric focusing followed by SDS-PAGE) showed that salt-stress induced a number of new proteins and repressed several others.

The study of readily measurable agronomic traits which may estimate total dry matter production and the determination of their relationships with quality characteristics needs more emphasis in alfalfa breeding. The main objective of this study was the selection of specific stem weight (SSW) and its relationship to other agronomic and nutritive traits. A secondary study was on agronomic and nutritive traits and their relationship with one another. Means, standard deviations, analyses of variance, and correlations were obtained on 98 Mesa-Sirsa alfalfa clones for SSW, plant height, yield, leaf-stem ratio, stem acid detergent fiber (ADF), leaf ADF, stem acid detergent lignin (ADL), and leaf ADL. The means of SSW (70-47 mg/5 cm), plant height (58-48 cm), yield (52-23 g), leaf-stem ratio (2.1-1.0), stem ADF (45-42%), and stem ADL (10.1-9.1%) varied from one harvest to another depending on the temperature. Significant differences were observed among alfalfa clones on June 1980 for SSW, plant height, yield, leaf-stem ratio, stem ADF, stem ADL, and leaf ADL. Range values were as follows: SSW (92-53 mg/5 cm), plant height (70-41 cm), yield (82-22 g), leaf-stem ratio (1.6-0.6), stem ADF (50-36%) stem ADL (13-8%), leaf ADF (18-13%), and leaf ADL (5-3%). A low correlation was reported between stem ADF and SSW (0.34) and also was observed between stem ADF and leaf ADF (0.46). The SSW probably would be more effective in increasing total dry matter production in dense stands. The coarseness of the stem was not related with high fiber. High leaf ADF could be useful in selecting for leaf cells which are more resistant to rupture and would gradually release soluble protein in the rumen; thus, the incidence of bloat probably would be reduced. A low stem ADF would make the stem more digestible; therefore, intake of the stem fraction may be greater. With more coarse material in the rumen, salivary secretion might be stimulated. The SSW and stem ADF could be used to select individual genotypes which could increase growth and development and at the same time improve forage quality, since SSW and stem ADF are not related.

Cold-acclimation-specific (CAS) gene expression was examined by screening a cDNA library prepared from poly(A)$ sp+$ RNA of cold-acclimated seedlings of a freezing-tolerant variety of alfalfa (Medicago falcata cv Anik). Three distinct CAS cDNA clones, pSM784, pSM2201, and pSM2358 were isolated. The genes corresponding to all three clones are coordinately induced by cold. Expression of these genes is not triggered by other stress treatments such as heat shock, water stress, wounding, or treatment with exogenous ABA. A positive correlation was observed between the level of expression of each gene and the degree of freezing tolerance of four alfalfa cultivars. / A full-length cDNA clone for the most abundantly-expressed gene, cas18 was isolated and sequenced. The deduced polypeptide, CAS18, is relatively small (167 amino acids), is highly hydrophillic, rich in glycine and threonine, and contains two distinctive repeat elements. It exhibits homology with members of the LEA/RAB/Dehydrin gene family--proteins which accumulate in response to water stress or abscisic acid (ABA). The cas18 cDNA hybridizes to three transcripts of 1.6, 1.4 and 1.0 kb in cold acclimated seedlings and cell cultures. The clone described here, Acs784, corresponds to the 1.0 kb transcript. / Expression of this gene is 30-fold greater in cold-acclimated cells than in nonacclimated cells after one week of low temperature treatment. Return to room temperature (deacclimation) results in the rapid disappearance of the three transcripts within just 5 hours. Studies of nuclear "run-on" transcription and transcript stability show that low temperature regulates the expression of cas18 at both the transcriptional and post-transcriptional levels.

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