A transgenic system for the heterologous expression of bioproducts in soybean (Glycine max (L.) Merrill) seed coats was developed. Expression was driven by the region upstream of the high expressing, seed coat-specific Ep gene, which encodes soybean peroxidase (SBP). Genes were modified to include sequences encoding the N- and C-terminal propeptides from SBP, which were predicted to target proteins to the vacuole. To investigate the influence of targeting on foreign protein accumulation, a model system was developed. Arabidopsis thaliana (L.) Heynh. plants were transformed with genes encoding SBP-green fluorescent protein (GFP) fusions, targeted to the cytoplasm, apoplast, endoplasmic reticulum (ER), or vacuole with combinations of the SBP N- and C-terminal propeptides and an ER retention signal. The location of the targeted fusion proteins and the function of the SBP N- and C-terminal propeptides were confirmed. Average specific activity of all three of the targeted fusion proteins was significantly greater than the average specific activity of the non-targeted, cytoplasmic fusion protein. Immunoblotting revealed that the GFP portion of the fusion protein was being partially degraded, and that degradation was more severe during targeting to the vacuole and apoplast than the ER. These results suggest that targeting proteins to any of the three compartments investigated can significantly improve protein accumulation but that maximum stability may be found in the ER. To compare the ability of soybean seed coats to express foreign proteins at levels comparable to the Ep gene, the Ep cDNA was heterologously expressed in a soybean cultivar that contains a deletion in the Ep gene, designated epep. Transgenic Ep plants produced SBP but activity levels were lower than those measured in the EpEp cultivar Harosoy 63. This suggests that elements necessary for the high expression levels of the Ep locus were omitted in the construct. To investigate the applicability of metabolically engineering soybean seed coats to divert metabolism towards the production of novel biochemicals, we introduced the polyhydroxybutyric acid (PHB) biosynthetic enzymes into soybean. PHB was produced at levels up to an average of 0.13% of seed coat DW. Although the levels of PHB are low, these results demonstrate that it is possible to metabolically engineer soybean seed coats.

This research was conducted to determine the potential for using plants to obtain samples of boron isotopes from groundwater, sod moisture and fracture water. In essence, this work sought to ascertain whether plants can function as in-situ samplers for boron as an environmental isotope. At present, very little is known about the behavior of boron isotopes in plants so this study was designed to reveal any isotopic fractionation that might occur during plant uptake by a specific species under carefully controlled conditions. The relationship between the boron isotope ratios sequestered in the leaves of the species Atriplex canescens and the growth conditions of the plant were investigated using a semi-hydroponic greenhouse experiment. Nutrient boron concentration and solution pH were selected as experimental variables as these parameters span large ranges in nature. In addition, the mechanism through which plants take up anionic nutrients suggests that boron isotope fractionation could occur. The experimental setup was a randomized factorial block design and the plants were provided six different nutrient solutions with pH values ranging from 7.5 to 9.5 and boron concentrations varying from 0.1 mg/L to 10.0 mg/L. Boron concentration in the plant's leaf and stem samples followed expected patterns, with the highest boron amount in the leaves of the plants fed nutrient solution with 10.0 mg/L B. The stern samples of plants fed 0.1 mg/L B contained the least boron. The ratio of boron in plants fed 10.0 mg/L vs. 0.1 mg/L B was far less than the 100:1 ratio of boron in the nutrients, which implies that a component of uptake is actively controlled by the plant. Negative thermal ionization mass spectrometry was employed to analyze the minute amounts of boron extracted from digests of the plant tissues. Statistical tests were utilized to determine that, contrary to the hypothesis, no significant isotopic fractionation occurred during uptake at any treatment pH level. The results of this research indicate that the species Atriplex canescens can provide samples of boron isotopes which closely represent the isotopic signature of the plant's water source.

Protoplasts were isolated from mesophyll tissue, callus and seedling cotyledons of Medicago sativa L. cv. Regen S and the halophyte M. marina L. Cotyledon protoplasts of Regen S were cultured in protoplast and cell culture media used previously for alfalfa protoplast culture and in media that had been simplified. There were no differences in the plating efficiencies of protoplasts cultured in the simple and complex media, but cells produced in the latter were greener and they colonized sooner. Protoplasts of M. marina grew at one-half the rate of Regen S protoplasts. Etiolated cotyledon protoplasts of Regen S were fused at 31°C using a solution containing PEG, DMSO and calcium at high pH. The frequency of fusion was 16% of the surviving protoplasts. These methods for protoplast isolation, culture and fusion should be useful in the somatic hybridization of alfalfa and M. marina.

Nine physically and chemically different soil samples from five study sites in which agaves grew, or were grown, were investigated to evaluate the effects of soil physical and chemical properties on the growth habits of agaves. In five Arizona study sites, biomass data of seven agave species has been recorded for the past ten years. Agaves were grown experimentally in the greenhouse using two widely different soil types from the five sites to evaluate growth under controlled conditions. Influence of edaphic factors on agave growth for the study sites and greenhouse experiment was evaluated. The results show that the determinant primary factors were water availability and temperature. Soil texture, soil pH, soil CO2 concentration, nitrogen, and soluble salt concentration were placed as influential secondary factors for the growth of agave. The degree of influence of these soil factors depends highly upon the genetic characteristics of agave species.

In April 1989, lettuce (Lactuca sativa L.) plants showing severe root rot symptoms were received for diagnosis from a commercial hydroponic facility in Southern California. A species of Phytophthora was consistently isolated from necrotic roots. Pathogenicity trials were conducted under hydroponic conditions in a greenhouse at root temperatures of 18 and 28 C. Root necrosis, along with stem decay and plant death, occurred within 5-7 days after inoculation at both temperatures. Reisolation of the fungus from roots and stems of inoculated, but not from roots of non-inoculated plants, confirmed pathogenicity. The fungus was identified as Phytophthora cryptogea Pethybr. and Lafferty on the basis of cultural and morphological characteristics. However, crosses with A1 and A2 mating types of Phytophthora cryptogea and other species of Phytophthora were not successful. This is the first report of Phytophthora as a root pathogen of cultivated lettuce.

Guayule (Parthenium argentatum Gray), a semidesert shrub was evaluated as a potential rubber crop. Since polyploid guayule reproduces by apomixis, progeny of individual plants should duplicate the characteristics of their parent. This study was conducted to estimate variations within and among progeny families from single-plant selections. Ten progeny per family were individually evaluated for plant height, width, fresh and dry weight of clipped branches, rubber and resin content, and yield. Leaves of selected plants were analyzed by gradient polyacrylamide gel electrophoresis for esterase and peroxidase to estimate genetic relatedness between parents and progenies. Negative correlation was between parents and progenies for most characters, but none were statistically significant from zero. This suggests a low heritability for the characters measured. Esterase and peroxidase detected variation in banding between parents and progenies and among progenies. This study suggested that out-crossing and meiotic reduction have occurred among apomictic guayule lines.

Since the mid-1970's, there has been active research on the development of Vernonia galamensis (Cass.) Less. as a potential new oilseed crop. Vernolic acid (cis-12:13-epoxy-cis-9-octadecenoic acid) comprises 70--75% of vernonia oil and is chemically reactive, affording it a variety of industrial applications. A concern in the domestication of an oilseed crop is to establish a breeding program to improve oil quality traits. The objectives of this research were to (1) develop a rapid procedure for seed analyses; (2) determine the development of vernonia oil; and (3) estimate the narrow-sense heritability (h 2) of oil quality traits. Successful separation of free fatty acids (FFA) and acylglycerides from small vernonia seed samples was achieved using aminopropyl solid phase extraction columns. Acylglycerides were eluted with a mixture of chloroform and isopropanol, while FFA were eluted with a mixture of acetone and trifluoroacetic acid. Four breeding lines from a collection of Vernonia galamensis held at the US Water Conservation Laboratory in Phoenix, AZ were used for the oil development study and grown in field trails in Phoenix and Tucson, Arizona. Seeds were collected on nine different days after flowering over the course of seed maturation. Seed samples were analyzed for FFA, acylglyceride constituent fatty acids, total acylglycerides, and total oil. In each breeding line, FFA content changed significantly throughout the course of the measurement period, and synthesis of acylglycerides constituent fatty acids followed a previously described pathway proceeding from C16:0 to C18:0 to C18:1 to C18:2 to C18:1 epoxy. Vernolic acid increased late in the measurement period, while total acylglycerides and total oil increased steadily over the period. Mature vernonia seed exhibited substantial variation in the amount of FFA, acylglyceride constituent fatty acids, total acylglycerides, and total oil. Sixty-nine half-sib families were created to study the heritability of FFA, vernolic acid, acylglycerides, and total oil production. Mature capitula were collected and analyzed individually for oil constituents. Narrow sense heritability estimates for these four oil quality traits were: FFA = 33%, vernolic acid = 65%, acylglycerides = 47%, and total oil = 50%. The results indicate potential for progress in selection for these traits.

Studies were performed to determine the concentration of soluble carbohydrates and starch in chrysanthemum plants at various stages of vegetative and floral development. Leaves of pot chrysanthemum (Chrysanthemum morifolium 'Charm' or 'Favor') grown under full irradiance had more soluble carbohydrates and starch than those grown with 65% irradiance reduction. Both showed clear diurnal variation in carbohydrate concentrations. Inflorescences exhibited no diurnal fluctuations in total soluble carbohydrate (TSC). Sucrose was the only translocated carbohydrate in chrysanthemums in quantities detectable by HPLC. In a postproduction environment, leaf and stem TSC remained relatively unchanged while inflorescence TSC decreased significantly. Reducing sugars (glucose + fructose) accounted for up to 84% of the inflorescence TSC. Relative levels of starch and fructans over time suggests an alternate use of fructans and starch as pools of available reserve carbohydrate during floral development. Fructans were shown to decrease in polymerization in both petals and inflorescences as petals expanded.

This study evaluated the success of recently revegetated roadsides in Tucson, Arizona. The primary objectives of this study were to (1) evaluate a sample of revegetated roadsides by assessing site condition and comparing existing vegetation to the original seed mix; and (2) develop recommendations for appropriate revegetation practices for use in Tucson. Density data were collected at 20 locations using belt transects to derive species information such as origin, vegetative life-form, and invasiveness. Results indicated that the study sites had greater proportions of plant material not specified in the original construction documents than specified plant material. Of the specified species, creosote bush, desert senna, and triangle-leaf bursage had the highest rates of establishment while brittlebush, globemallow, shrubby buckwheat, and fourwing saltbush provided the most vegetative cover. Several non-specified species exhibited successful establishment on many study sites, while some species rarely or never appeared in the study area despite frequent specification.

C. roseus hairy roots cultures were employed to prove the ability of plants to take up and transform TNT in the absence of microbes. Mass balances performed with 14C-TNT showed that the TNT was being taken up and transformed and not mineralized. The transformation pathway was seen to be at least initially reductive. However, in cases where no TNT was detectable at experiment's end, standards could only account for 5% of the final products. Pseudo-first order reaction kinetics were observed for low initial concentrations of TNT in both stationary and exponential phase hairy root cultures. Kinetic constants ranged from 0.0103 to 0.0161 L/g-day for stationary phase experiments performed with 15 to 35 mg/L initial TNT concentrations. Kinetic constants of 0.0075 to 0.0156 L/g-day were observed for experiments performed on exponential growth phase hairy roots with an initial TNT concentration of 20 to 40 mg/L. Toxic effects were seen at all phases of growth and all concentrations measured.