Mountain Scholar :: Browsing by Author "Hughes, Harrison G., advisor"

Browsing by Author "Hughes, Harrison G., advisor"

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Open Access
Asexual propagation of blackbrush (Coleogyne ramosissima Torr.)
(Colorado State University. Libraries, 1990) Weglinski, Eugene, author; Hughes, Harrison G., advisor; Redente, Edward F., committee member; Orr, Greg L., committee member
Mining disturbances in Canyonlands National Park occur, in part, in monotypic stands of blackbrush (Coleogyne ramosissima Torr.). Blackbrush does not readily reseed itself following disturbance, therefore, stem cuttings and mound layering were evaluated as methods for asexual propagation as a means of providing plants to be used in a revegetation program. Rooted cuttings were planted to evaluate their use in revegetation. Rooting of stem cuttings was evaluated using talc, 0.3% (w/v) indole-3-butyric acid (IBA), 0.8% (w/v) IBA, and Rootone, a commercially available rooting hormone mixture. Treatments were applied to cuttings collected from new, one-year-old, and older wood (2+ years) at three separate dates. Propagation by stem cutting proved successful with highest rooting percentages achieved using current year's growth with application of supplemental rooting hormone. Differences between hormone treatments were insignificant. However, differences were found in comparisons between hormone treatments and a talc control. Mound layering was investigated with 20 plants at each of three sites and involved removal of all growth over 2.5 cm above ground level. Supplemental water was applied to half the plants for the duration of the study. Plants were buried to one-half of the height of new growth on a monthly basis. Plants responded to the treatment with a flush of growth but did not root. Thirty-eight cuttings rooted the prior year were planted in spring 1989 in an abandoned roadbed to evaluate field establishment. Treatments included application of supplemental water at two week intervals. Results were inconclusive.
Open Access
Characterization of Alstroemeria species and cultivars using random amplified polymorphic DNA (RAPD) analysis
(Colorado State University. Libraries, 1997) Picton, Deric D., author; Hughes, Harrison G., advisor; Ward, Sarah, committee member; Stushnoff, Cecil, committee member
The characterization of Alstroemeria has not been effective through the use of conventional morphological markers. This is mainly due to the confused status of many of the species. Breeding of Alstroemeria has involved interspecific crosses as well as the use of mutagens and chromosome doubling for the creation of cultivars. Through the use of molecular techniques, in particular random amplified polymorphic DNA (RAPD) analysis, it is possible to uniquely characterize the species and cultivars. Fifteen species and hybrids along with 25 cultivars were examined using RAPD analysis. Four primers were eventually used for the analysis. All amplification products were separated using a 5% polyacrylamide gel and stained with silver nitrate for maximum differentiation of the fragments. The four primers yielded 73 amplification products which were polymorphic. When analysed using cluster analysis, all species and cultivars were uniquely characterized and putative parentages of many of the cultivars were determined.
Open Access
Characterization of chickpea (Cicer arietinum L.) accessions using molecular techniques
(Colorado State University. Libraries, 1998) Hamdaoui, Fatiha, author; Hughes, Harrison G., advisor; Johnson, Duane, committee member; Nabors, Murray, committee member; Towill, Leigh E., committee member
The productivity of chickpea has not been markedly improved through conventional breeding. The main problem for increasing yield is the susceptibility of the plant to the disease caused by the ascomycete Ascochyta rabiei. Because genetic markers may speed up chickpea breeding for resistance to ascochyta blight, isozymes and RAPD techniques have been applied to 56 chickpea germplasm lines which have been screened against ascochyta blight in the field and at the greenhouse in Morocco. Artificial inoculation at three locations, resulted in none of the lines evaluated being immune, seven entries were resistant and the remaining were tolerant or susceptible under Morocco conditions. Two hundred primers for RAPD assay and 15 enzymatic systems were assayed. The fifteen enzymatic systems tested were almost monomorphic and were not able to discriminate among the tested lines. Among the 200 primers tested only 6 primers yielded polymorphism. Forty- one amplification products were produced and among them four were associated with disease resistance to ascochyta blight. RAPD procedure with polyacrylamide gels differentiated among the resistant and susceptible cultivars and produced more polymorphisms than RAPD using agarose gels.
Open Access
Chromosome races and polypoid cytoforms in Distichlis spicata
(Colorado State University. Libraries, 2001) Reid, Scott D., author; Hughes, Harrison G., advisor; Koski, Anthony, advisor
Saltgrass (Distichlis spicata [L.] Greene) is a salt-tolerant C4 grass native to the Americas that is currently being evaluated for use as a turfgrass species. Saltgrass has a highly variable morphology in different regional occurrences, and this has led some botanists to consider it as two species or as comprising several botanical varieties over its wide distribution in the U.S. west of the Mississippi River and along coastal regions. Chromosome numbers of 160 saltgrass accessions, primarily from nine western states, were determined. A 38-chromosome saltgrass race with distributions separate from the previously known 40-chromosome type has been identified. Both races are fertile and hybridize readily. The distribution pattern and the cytological behavior of hybrids between the races suggest they are adaptively different. No aneuploidy in the 38-chromosome race has been found, and no deficient aneuploids have been found in the 40-chromosome race. Meiotic irregularities in the 40-chromosome race suggest a pathway for the evolution of the 38-chromosome race, dependant on hypothesized genetic changes that allow fitness in individuals lacking the pair of homologues defining the difference between the races. More research is needed to evaluate broad but varied morphological differences between the races. The consequences of intermating the two cytoraces in breeding efforts are unknown, but karyotypic instability may result in some cases. An octaploid saltgrass cytotype, previously considered rare, has been found to occur more commonly, distributed among both of the cytoraces. One accession, believed to be a 6x cytotype, represents a ploidy level previously undescribed in saltgrass. Screening to identify individuals of different ploidy levels among collections made for the turfgrass breeding program is advisable. Aneuploidy and multivalent associations in meiosis suggest the higher-order polyploid cytoforms may reproduce with less efficiency sexually, but this has not been adequately studied. Two ca. 74-chromosome accessions showed high estimated pollen viability. B chromosomes are reported for the first time in saltgrass, occurring in some individuals of all three cytotypes.
Open Access
Chromosome studies of Alstroemeria pelegrina L.
(Colorado State University. Libraries, 1990) Stephens, Janice L., author; Tsuchiya, Takami, advisor; Hughes, Harrison G., advisor; Lee, Chi Won, committee member
Karyotype analysis is one of the approaches to determine the parental species used for hybrid variety breeding. Alstroemeria pelegrina L. is considered to be one of the primary parents used in the development of many commercial cultivars of Alstroemeria. Somatic chromosomes were prepared from root tip cells using the acetocarmine squash method. Measurements from these cells were then used to develop a karyotype for A. pelegrina L.. The genome of A. pelegrina L. was shown to consist of two groups of four chromosomes each. The first group consists of metacentric or submetacentric chromosomes in which the smallest submetacentric is satellited. The second group consists of four acrocentric chromosomes in which chromosomes 5, 7 and 8 are satellited. The small submetacentric satellited chromosome in the first group provides a marker chromosome which is unique to A. pelegrina L.. The presence of this chromosome in the karyotype of a cultivar would indicate that A. pelegrina L. was a parent of that cultivar. Although A. pelegrina L. was shown to have a very similar karyotype for all the plants studied there were some differences between them. This indicates that chromosomal changes may be occurring within the species in individual plants resulting in karyotypic polymorphism. A study of the anthers from immature buds at anaphase I showed 8-8 separation without any abnormality. The stages of diakinesis or metaphase I showed that chromosome pairing was normal with 8 bivalents. Pollen viability varied from 44.7% in A. pelegrina L. 'rosea' to 80% in A. pelegrina L. 'alba'.
Open Access
Cold hardiness and cryopreservation of small fruits
(Colorado State University. Libraries, 1981) Wu, Min-Tze, author; Hughes, Harrison G., advisor; Wallner, Stephen J., committee member; Stanwood, Phillip C., committee member
This study examined cold hardiness and the occurrence of deep supercooling in stem with an attached bud of 4 cv. of grape and 4 of raspberry. Cryoprotectants were also tested for their influence on cold hardiness of raspberry vegetative bud and stem. Differential thermal analysis (DTA) of 4 cv. of grapes, Vi tis species hybrids, 'Concord', 'Beta', ' Valient', and 'Rougeon' showed that the stem and bud pieces of all cv. deep supercool in winter. They all showed a bud exotherm at approximately -25 to -30°C, and a stem tissue exotherm at approximately -40°C. The temperature at which the bud exotherm occurs may be the killing point of the bud which in turn could lead to the death of the above-ground canes. Evidence of this was the observation of the death of 'Rougeon' buds, bud exotherm observed at approximately -20°C, during the winter of 1980-1981 when the lowest temperature was -2S.6°C. DTA profiles of stem with attached bud samples of the 4 raspberry cv., 'Heritage', Ruhus idaeus L., 'Black Hawk', R. occidentalis L., ' Amethyst', R. neglectus Peck, and 'Darrow', R. allegheniensis Porter, suggest that these do not deep supercool during winter. The mechanism of freezing resistance of these may be tolerance of extracellular freezing. Survival, indicated by sprouting, on January 30, 1981 showed that the LT50 (lethal temperature for at least 50% bud sprouting) was -55°C -50°C, and -45°C for 'Black Hawk', 'Amethyst', and 'Heritage', respectively. This supports the idea that at least these 3 cv. resist freezing by tolerance of extracellular freezing. Therefore minimum temperature may not be the major limiting factor in their northernly distribution. In descending order the observed degree of hardiness was 'Black Hawk', 'Amethyst', and 'Heritage'. Survival tests of 'Heritage' samples treated with cryoprotectants showed no benefit of cryoprotectants for enhancing the resistance of acclimated buds and stem to cold injury. However, there were some implications that cryoprotectants may enhance the resistance of nonacclimated samples to lower temperatures.
Open Access
Cryopreservation of sweet potato shoot tips by vitrification
(Colorado State University. Libraries, 1997) Pennycooke, Joyce C., author; Hughes, Harrison G., advisor; Ward, Sarah, committee member; Towill, Leigh E., committee member
The conservation of vegetatively propagated germplasm is problematic. In vitro conservation of sweet potato has been achieved using normal and limited growth regimes. However, although the storage of in vitro plants is advantageous, it is not the method of choice for long term conservation because normal growth conditions require frequent subculturing which makes it labor intensive and costly. Also, frequent subculturing over a prolonged period of time may lead to undesirable consequences such as, contamination, selection and chromosomal aberrations. Cryopreservation offers the simplest and most economical way for the conservation of plant germplasm and vitrification is the preferred method to accomplish this. Previously, Towill and Jarret (1992) reported that vitrified sweet potato shoot tips exhibited callus formation and that the vitrification protocol developed included high levels of within and between treatment variability for explant survival. The purpose of this study was therefore to, examine the various steps of the cryopreservation procedure in order to generate a reproducible shoot tip cryopreservation protocol for sweet potato. In vitro grown sweet potato shoot tips were excised at 0, 3 or 10 hr in light after an 8 hr dark period. Excised shoot tips were precultured in 2% sucrose in MS for 24 hr and 0.3, 0.5 or 0.75 M sucrose in MS for an additional 24 hr. Precultured shoot tips were loaded with two different cryoprotective solutions for 20 or 60 min at 22 °C and then dehydrated with a concentrated vitrification solution (PVS2) for various lengths of time at 22 °C. Following dehydration, shoot tips were cooled by placing them along with a small drop of PVS2 on thin strips of aluminum foil, in 0.2 ml PVS2 in polypropylene straws or in 1 ml of PVS2 in cryo-vials. These were then plunged in nitrogen 'slush' (ca. -209 °C). Cryoprotectant-exposed shoot tips as well as vitrified shoot tips were recovered on MS media with various modifications (mineral composition, surfactant/ antioxidant, hormonal content). Another type of vitrification technique, the encapsulation / dehydration vitrification procedure, was also pursued for the cryopreservation of sweet potato shoot tips. The highest survival (67%) of vitrified encapsulated shoot tips was achieved after 4 hr dehydration corresponding to a moisture content of 18.1 %. To our knowledge, this is the first reported survival of cryopreservation of sweet potato shoot tips using the encapsulation / dehydration procedure. For the solution-based vitrification method, shoot tip survival after both the dehydration step and cooling in nitrogen slush was strongly dependent on the preculture condition (sucrose concentration). The best survival was achieved from shoot tips (PI 290657) excised from in vitro plants soon after the 8 hr dark period, with a 24 hr preculture in 0.3 M sucrose prior to loading in 2M glycerol + 0.4 M sucrose for 1 hr and then dehydrated with PVS2 for 16 min at 22 °C respectively. A fast cooling rate was beneficial and this was achieved by placing the shoot tips along with a small drop of PVS2 on thin strips of aluminum foil prior to plunging in nitrogen slush. Survival of cryopreserved shoot tips was promoted significantly (P = 0.0001) in the absence of NH4+ in the recovery medium for 5 days prior to transfer to regular MS medium. In this study, we report an improved vitrification protocol in that survival after dehydration and cooling results in the resumption of shoots initiated directly from treated shoot tips. Most important for any genebank is the regeneration of shoots so as to reduce the risk of somaclonal variation. Using the same recovery medium, for all four lines tested, relatively good survival levels (ranging from 62% - 97 %) were achieved, and most important, all lines regenerated shoots after cooling to cryogenic temperatures.
Open Access
Cytological and biochemical characterization of Alstroemeria, Leontochir and Bomarea
(Colorado State University. Libraries, 1995) Stephens, Janice L., author; Hughes, Harrison G., advisor
lsozyme characterization and cytological analysis were used to characterize and identify species and cultivars of Alstroemeria, Leontochir and Bomarea. Twenty four species, hybrids and color variants of Alstroemeria, two plants of Leontochir ovallei and one plant of Bomarea species, as well as 23 cultivars were available for isozyme characterization. Nine species of Alstroemeria, two Leontochir ovallei, one Bomarea species and 22 Alstroemeria cultivars were investigated to determine their karyotypes and the Giemsa banding patterns of their chromosomes. Seven isozyme systems were identified which exhibited a high level of polymorphism among the species. A single technique was developed for the extraction of all of these enzymes. The enzyme systems investigated were phosphogluco-mutase, phosphogluco-isomerase, 6-phosphogluconate dehydrogenase, aspartate amino transferase, malic enzyme, esterase and leucine amino peptidase. It was found that between 11 and 18 of the species and hybrids could be identified uniquely for each of the first six enzyme systems. The final system, leucine amino peptidase was tested on only 11 species and hybrids and 9 different patterns were identified. Using only 3 of the seven enzyme systems it was possible to uniquely identify all of the species and hybrids investigated. When the 23 cultivars were analyzed, using the first six enzymes, a high level of variability was evident. This was expected due to the polyploid nature of most of the cultivars. The highest level of variability was found for esterase with which 20 of the 23 cultivars exhibited unique banding patterns. All of the cultivars could be uniquely characterized using a combination of only two of the six enzymes. Very little is known about the parentage of most of the cultivars. Comparison of the banding patterns of the cultivars and the species allowed the identification of the possible parents of each of the cultivars studied. Using both karyotype analysis and Giemsa banding it was possible to identify individual chromosomes within each species. The identification of a particular species could then be described in terms of the characteristics of each chromosome pair within the complement. The karyotype of all three genera was quite similar, particularly the distribution of acrocentric and the smaller metacentric and submetacentric chromosomes. However, Alstroemeria had 2n = 2x = 16 chromosomes, whereas Leontochir and Bomarea had 2n = 2x = 18 chromosomes. Characterization and identification of cultivars was also possible using these techniques. Each cultivar studied had a unique complement of chromosomes which clearly identified the cultivar. Using these two approaches it is possible to uniquely identify the species and cultivars of all three genera. This information may now be used to determine the probable parentage of the cultivars. The present study also provides an approach which can be used to further the understanding of the relationships between these three genera. When more species have been studied and their unique characteristics revealed, it will be possible to use this information to clarify the identity of the putative parents of the European cultivars.
Open Access
Drip irrigation of plastic mulched strawberry using carbonated water - a greenhouse study
(Colorado State University. Libraries, 1994) Shore, Margaret L., author; Hughes, Harrison G., advisor; Smith, D. H., committee member; Moore, Frank D., III, committee member
Carbonated water irrigation enhanced yields of tomato, however, little is known about the mechanism of this response. Objectives were: 1.) determine if strawberry responds to irrigation with carbonated water and 2.) determine if yield increase, should it occur, is due to short-term soil pH optimization or air-soil atmospheric enrichment with CO2. Two different soils (2:1, perlite:soil) were used: a calcareous soil (5% CaCO3, pH 8.0), with a Zn content of 0.9 μg/g and a non-calcareous soil (< 1 % CaCO3, pH 6.4) with a Zn content of 8.4 μg/g. The carbonated water temporarily lowered the pH of the calcareous soil to 6.7 and the non-calcareous soil to 5.9, at both extremes of the optimal range (5.2-6.4) for strawberry. There was significant increase in above ground (1 cm) CO2 during irrigation. Also, a significant increase in soil CO2 was observed in the calcareous soil, carbonated water treatment over the noncalcareous, carbonated water treatment, which suggests carbonic acid played a role in lowering the surface pH of the calcareous soil from 8.0 to 6.7 shortly after each irrigation event. Application of carbonated water increased production of buds and open flowers at the P < 0.05 significance level. Carbonated water increased the production of marketable fruit (P < 0.10) as compared to the noncarbonated water considering both soils. In addition, there was greater crown dry weight and higher leaf chlorophyll content (P < 0.05) observed in plants irrigated with carbonated water. The magnitude of the response to carbonated water was similar for each soil. The noncalcareous soil had significantly greater accumulation of Zn in leaf tissue as compared to calcareous soil, considering both irrigation treatments. However, the calcareous soil, carbonated water irrigation treatment had a slight increase in the uptake of Zn over the calcareous, noncarbonated water treatment. Also, there was no significant difference in the uptake of Fe, Mn, or Cu in regard to irrigation treatments or soil type.
Open Access
Environmental stress aspects of saltgrass [Distichlis spicata (L.) Greene]
(Colorado State University. Libraries, 2002) Shahba, Mohamed Ahmed, author; Qian, Yaling, advisor; Hughes, Harrison G., advisor; Wallner, Steven J., committee member; Brick, Mark A., committee member
Saltgrass [Distichlis spicata (L.) Greene] is undergoing preliminary evaluation at Colorado State University for use as a turfgrass in adverse environments. Furthermore, it has a potential as a range species in saline-alkali basins and many of the salt marsh areas in addition to its importance for wildlife. In cooperation with a saltgrass breeding project, the objectives of this dissertation work were to: (a) determine freezing tolerance of saltgrass; (b) determine the relationship between freezing tolerance and nonstructural carbohydrate content; and (c) determine nitrogen requirements and to evaluate the nutritive value of saltgrass as affected by N levels. Stolons of saltgrass accessions were sampled at monthly intervals from October 1999 to April 2000 and from October 2000 through April 2001 and subjected to laboratory freezing tests. Parts of the sampled stolons were used to assess soluble carbohydrates, including sucrose, fructose, glucose, raffinose and stachyose using gas chromatography (GC). Results indicated significant differences among accessions in LT50 (subfreezing temperature resulting in 50% mortality) and carbohydrate content. Ranking of accessions for LT50 (°C) during January, 2000 was A29 = 48 (-20.0) > 55 (-17.0) ≥ 32 (-15.5) ≥ A65 = C66 (-14.0). In January, 2001 they were ranked with 48 = 55 (-26.0) > A65 = 32 (-23.0) > A29 (-20.0) = C66 (-18.5). Sucrose was the predominant sugar, but did not show a clear seasonal trend and had no correlation with freezing tolerance. Fructose, glucose, raffinose and stachyose exhibited clear seasonal changes, showing highest concentrations during mid-winter. Higher fructose, glucose, or raffinose concentrations were frequently observed in accessions 48, 55, and A29, which coincided with their lowest LT50. In contrast, C66 had the lowest sugar concentrations overall, which related to its sensitivity to lower temperatures. Accessions A24 and A138 were planted in the field at the Horticulture Research Center, Fort Collins, CO. to determine the nitrogen requirements for these accessions during establishment and to evaluate their nutrient content as related to nitrogen level. Results indicated positive linear relationships between cover %, productivity, tissue nitrogen and protein contents with applied nitrogen levels in both seasons. Ca, P and Fe had a positive association while Na, S and Mg had a negative association with nitrogen levels. Establishment in terms of cover and productivity, and nutritive value of the two tested saltgrass accessions increased with increasing N fertilization rate. However, nitrogen had no effect during the first month of establishment on cover when water was critical. Plots which received total of 450 kg/ha showed the best cover percentage.
Open Access
Evaluation of plant characteristics and disease resistance in Cu-ipt transformed watermelon cv. crimson sweet
(Colorado State University. Libraries, 2004) Goktepe, Fahrettin, author; Hughes, Harrison G., advisor; Byrne, Patrick F., committee member; Vivanco, Jorge M., committee member; Hill, Joseph P., committee member
Watermelon cv. Crimson Sweet was transformed with the copper inducible isopentenyl transferase gene (Cu-ipt) via Agrobacterium mediated gene transformation process. The ipt gene governs the rate-limiting step in the cytokinin biosynthesis pathway. The transformants were confirmed via polymerase chain reaction (PCR) in the plant with ipt specific primers. Cu-ipt transformed plants were treated with copper sulfate at a concentration of 5, 10, or 50 μM copper sulfate to determine if the gene could be activated by copper at three levels. Transformed plants treated with copper sulfate differed in evaluated horticultural characteristics from those non-transformed as well as transformed plants not sprayed with copper sulfate. Delayed leaf senescence, increased chlorophyll content, reduced apical dominancy and released axillary buds were significantly different in Cu-ipt transformants compared to non-transformant plants. Significant reduction of seed number in watermelon fruit was also observed in copper sulfate treated Cu-ipt plants as compared to the non-transformant plants. Other than some slight alterations, elevated endogenous cytokinin level didn't cause major interference with transformants normal growth and development. The application of copper sulfate also induced resistance against Gummy Stem Blight disease in Cu-ipt transformants and their seedlings compared to the non-transformant plants.
Open Access
Hydroponic screening of strawberry for salt tolerance: correlation with in vitro evaluations
(Colorado State University. Libraries, 1994) Wright, Floyd Thomas, author; Hughes, Harrison G., advisor
Strawberries (Fragaria x ananassa), as well as most agricultural crops, display sensitivity to increased salt concentrations in irrigation water and soil. Efforts to breed more tolerant crops have not been entirely successful in the past because many complicated factors are involved, but promising efforts continue. 'Fern' (F) and 'Douglas' (D) strawberries, two salt sensitive cultivars, were crossed with a salt tolerant beach strawberry selection, Fragaria chiloensis (C). Samples of the two crosses and cultivar parents were previously tested in 1989 and 1990 for salt tolerance in vitro as germinated seedlings. In 1991 and 1992, samples of the crosses and all parental types were hydroponically tested as mature plants for salt tolerance. The in vitro and hydroponic findings were correlated. Significant salt tolerance was noted in the 'Fern' X L. chiloensis crosses both in vitro and in hydroponic testing, although the growth response was different between the two methods. All genotypes tested in vitro grew better at the 0.2% salt concentrations when compared to control. At higher concentrations, there were varying degrees of growth reduction depending on the genotype. Two types of hydroponic experiments were run. In type 1, salts were added in 0.3% increments over time in order to determine the highest total concentration each plant genotype could tolerate given time to adapt by metabolic alteration. In type 2, salt was added in one application at 0.2%, 0.5%, or 0.8% NaCl in order to determine tolerance to an osmotic shock. Differences in relative growth were noted between the two hydroponic experiments. When lines were directly subjected to high salt, most plants were killed at the 0.5% level, and all plants were killed at the 0.8% NaCl level. When salt was gradually added, 1.3% was tolerated by the best adapted crosses. Despite the differences in tolerance noted, the genotypes tested ranked similarly under both systems; F X C was superior to D X C, and both crosses were superior to the cultivars. Therefore, the hydroponic results validate the in vitro results.
Open Access
Influence of storage temperature and time in storage on pigment content of potato (Solanum tuberosum L.)
(Colorado State University. Libraries, 2005) Alenazi, Mekhled M., author; Hughes, Harrison G., advisor; Holm, David G., advisor
Anthocyanins are responsible for different colors in many plant species including potato tubers. They are also one of the important components in human health as antioxidants. Important factors that can affect anthocyanin concentration in potato tubers are storage temperature and the length of storage. The influence of storage temperature and time in storage on tuber anthocyanin concentration was investigated in seven potato genotypes. These genotypes were cultivars All Blue and Yukon Gold as well as VC0967-SR/Y, Purple Majesty (CO9I6S-3P/P), Mountain Rose (CO94183-1R/R), VC1002-3W/Y and CO97232-2R/Y. Tubers of the seven genotypes were stored at 4°C and 10°C for 0, 4, 6, 8, 10, 12, 16, 20 and 24 weeks . Both fresh and freeze-dried samples of the tubers were evaluated for each of the temperature and time treatment combinations. Extractable anthocyanins were obtained in only three genotypes, CO94183-1R/R, CO94165-3P/P and "All Blue", as determined by the UV/Vis Molecular Device Spectra Max Plus 384 spectrophotometer. There was an increase in anthocyanin concentration with increased time in storage for both fresh and freeze-dried samples (P<0.0001). However, tubers stored in the cooler at (4°C) had much higher levels of anthocyanin than those tubers stored at 10°C. Increased levels of anthocyanins in cold-stored tubers are likely associated with the conversion of starch to sugar (so called "cold sweetening") and subsequent conversion to anthocyanins. More anthocyanins were extracted from freeze-dried tuber samples than fresh samples. Both techniques exhibited a similar trend in that an increase in length of time in storage had a similar level of increase in anthocyanin concentration. Extraction of anthocyanins from freeze-dried tissue is more efficient and effective than using fresh tissue for evaluation of anthocyanin concentration in potato tubers.
Open Access
Inheritance of cold stress tolerance in grapes
(Colorado State University. Libraries, 1989) Esensee, Virgil D., author; Hughes, Harrison G., advisor; Johnson, Duane, committee member; Lee, Chi Won, committee member; Towill, Leigh E., committee member
Cold hardy 'Valiant' was crossed to the lesser hardy cultivars 'Zinfandel' and 'French Colombard' to produce progeny that were later planted in the field for evaluation. In January and February, 1989, buds with subjacent stem tissue were evaluated by differential thermal analysis (DTA). The results were compared with those obtained from tests of browning that were performed following controlled freezing of buds in a methanol bath. DTA was found to be an accurate means to determine the mean midwinter killing temperature of grape buds. The apparatus used cooled the experimental tissues at a smooth linear rate (-15.8 C/hr) and produced low variances within replicates. An analysis of variance indicated that there were true differences between progeny means. Field evaluations in June, 1989, also verified the midwinter DTA data. These tests substantiate recommending DTA as a breeding tool for evaluating midwinter grape cold hardiness. Broad sense heritability was calculated using grape bud DTA data for both 'Valiant'x'Zinfandel' and 'Valiant'x'French Colombard' crosses. All calculations produced heritabilities near unity, indicating that of the progeny tested, the phenotypes were due primarily to expression of the genotypes. It was also found that poor seed germination commonly found in grapes can be ameliorated through the use of embryo rescue techniques. Data showed that low germination of grape seeds is not necessarily due to inviability, since rescued embryos grew and developed when placed on Nitsch's medium.
Open Access
Overcoming seed dormancy in Glaucium Spp. (Papaveraceae)
(Colorado State University. Libraries, 2007) Elsner, Karen C., author; Hughes, Harrison G., advisor; Kelaidis, Panayoti, committee member; Fenwick, Jack, committee member
The Horned Poppy, Glaucium sp., is a potential candidate for introduction as an herbaceous perennial to Colorado. However, its introduction has been hindered by poor germination, less than 10% in informal studies across the state. Nine seed lots of Glaucium sp., representing four different species and a hybrid harvested from four different year, were collected at Denver Botanic Gardens and used in research to develop a protocol for seed treatment to improve germination. Four different trials were conducted to evaluate germination. The first trial evaluated stratification temperatures and germination with and without light on two collection years of G. flavum. The greatest germination was 69.5% for the seeds collected in 2005 that were stratified for 45 days at 7°C and germinated with light. The seeds harvested in 2003 had 53 % germination with 7°C stratification for 45 days and germinated without light. The second trial evaluated scarification treatments, hot water, concentrated sulfuric acid (2 levels) and nicks in the seed coat, on two collection years of G. flavum and G. acutidentatum. 2005 G. flavum had the greatest germination at 57 % with a 30- minute sulfuric acid scarification. 2003 G. flavum had 20% germination with the 60- minute acid scarification. G. acutidentatum seeds from 2003 and 2005 germinated with acid treatments but not as well as G. flavum. 2003 G. acutidentatum had 6% germination with 60-minute acid scarification and 2005 G. acutidentatum had 1.5% germination with 30-minute acid scarification. Stratification and scarification were evaluated together in the third trial. Additional seed lots; 2003 G. grandiflorum, 2003 G. corniculatum and 1999 G. corniculatum x flavum were included. Although each seed lot had varying responses to the treatments, 30-minute acid scarification combined with 30- or 45-day stratification at 8°C were the optimal pretreatments. The germination percentages for these treatments ranged from 60 to 92%. The final trial compared gibberellic acid to stratification to determine if GA could substitute for cold stratification. Hydrogen peroxide was also evaluated for comparison purposes. For all the seed lots, 30-minute acid scarification with 400 ppm or 500 ppm GA3 generated the greatest germination. The percentages for these treatments ranged from 58 to 95%. Gibberellic acid proved to be a substitute for cold stratification; however, seeds germinated slower than cold stratification. Germination with GA took approximately 10 days while stratified seed germinated in five days. G. grandiflorum and G. acutidentatum can be considered to have intermediate complex morphophysiological dormancy with a hard seed coat, while G. corniculatum appears to have only a hard seed coat.
Open Access
Phenotypic variability, cold hardiness and flowering induction of saltgrass [Distichlis spicata (L.) Greene] clones
(Colorado State University. Libraries, 2006) Rukavina, Hrvoje Harry, author; Hughes, Harrison G., advisor; Brick, Mark A., committee member; Panella, Lee, committee member; Qian, Yaling, committee member
With increased population growth and periodic droughts in the semiarid U.S. west, there is interest in developing alternative turfgrass species that are water efficient and tolerate poor quality water. Colorado State University is currently evaluating saltgrass for its potential use as turf. Development of a new turfgrass cultivar requires an understanding of environmental factors that influence traits important for turf quality. Furthermore, cultivar development for northern climates and transition zones necessitates an understanding of cold hardiness. Finally, to enable hybridization throughout the year and make rapid progress in a breeding program, it is important to develop techniques and understand environmental stimuli that induce flowering in saltgrass. This study was initiated to: 1) characterize variation in morphological traits and time of leaf browning in fall among saltgrass clones relative to geographic and climatic variables at the location of clones' origin; 2) examine the relative freezing tolerance of saltgrass clones as related to climatic zone of origin as well as relationship between freezing tolerance and time of leaf browning in fall and 3) examine the influence of sampling time from the field, as well as burning and nitrogen fertilization on flowering induction of saltgrass clones from different environments. In the first experiment, traits of growth (morphology) and time of leaf browning in fall were measured on 53 saltgrass clones from 42 locations established at one location in Fort Collins, CO. Principal component analysis on the traits of plant morphology extracted the first principal component (PC-1) that explained 78% of variability and was used as the estimate of growth. Principal component analysis was followed by multiple regression of PC-1 and time of leaf browning in fall on the environmental variables at locations of clones' origin. Variation in saltgrass growth (morphology) was related to the seasonal climatic variables of summer drying and fall cooling that explained 50% of variability of morphological traits. Variation in time of leaf browning in fall was related to longitude and minimum winter temperature which together explained 60% of total variability of this trait. In the second experiment, rhizomes were sampled during 2004 and 2005 midwinters from 27 saltgrass clones from three cold hardiness zones established in Fort Collins, CO and then subjected to a freezing test. Saltgrass freezing tolerance was highly influenced by the climatic zone of clone origin in both years of the experiment. Clones with greater freezing tolerance turned brown earlier in fall in both seasons. This study indicated that saltgrass clones from northern (cooler) climates had greater freezing tolerance than clones from southern (warmer) areas. In the first year of the third experiment, three clones (A1540 from Colorado, 1490 from South Dakota and C1660 from Nevada) were sampled from the field twice (in August and November). In the second year, two additional clones from the Colorado Front Range (A1180 and A1610) and an additional sampling time (January) were included. In the first year, nitrogen fertilization increased number of spikes in saltgrass. Compared with August sampling, sampling in November increased the number of spikes, and had a greater effect on clone A1540 than on clone 1490. The burning treatment increased number of spikes only in plants sampled from the field in August. In the second year, nitrogen fertilization increased the number of spikes to a greater extent when nitrogen was applied with burning than without the burning treatment. In comparison with August sampling, sampling in November increased number of spikes in all clones with the greatest effect in clone A1540. Sampling in January additionally increased number of spikes in clones 1490 and C1610 without a significant effect on number of spikes in clone A1540. Burning treatment had its greatest effect on number of spikes in plants sampled in August, as compared with November and January sampling.
Open Access
Physical and chemical alteration of Distichlis spicata L. cell walls with NaCl and water stress
(Colorado State University. Libraries, 1991) Lopez-Gutierrez, Francisco, author; Hughes, Harrison G., advisor; McNeil, Michael, committee member; Ross, Cleon W., committee member; Towill, Leigh E., committee member
Cultured cells of the halophytic grass Distichlis spicata adapted to grow in 500 mM NaCl (-25 bar) exhibited an altered growth physiology that resulted in slightly slower cell expansion and fully expanded cells with volumes only one-half to one-third those of unadapted cells. The reduced cell volume occurred despite maintenance of turgor pressures sometimes 2-fold higher than those of unadapted cells. Tensile strength as measured by a nitrogen gas decompression technique showed empirically that walls of NaCl-adapted cells were weaker than those of unadapted cells. Correlated with this weakening was a substantial change in the organization of the matrix polysaccharides; specifically, glucuronoarabinoxylan was more readily extractable, perhaps caused by a decrease in cross-linkages with phenol substances. Despite a 3-fold decrease in the amount of hydroxyproline and approximately a 2-fold increase in tyrosine, the quantity of insoluble protein and, the proportions of crystalline cellulose remained relative constant. Glycosidic linkage analysis of carbohydrates revealed little modification in the quantity and primary structure of the matrix polysaccharides as a result of NaCl (salinity) and polyethylene glycol 8000 (water deficit) stress. The study revealed that changes in the superstructure of glucuronoarabinoxylan are the primary determinant in the tensile strength of NaCl-adapted cells.
Open Access
Physiological studies in acclimatization of in vitro tobacco plantlets
(Colorado State University. Libraries, 1992) Safadi, Farida, author; Hughes, Harrison G., advisor; Morgan, Jack A., committee member; Towill, Leigh, committee member; Nabors, Murray, committee member
Two acclimatization methods of in vitro tobacco (Nicotiana tabaccum 'Wisconsin 38') plantlets (IVP) were studied for their physiological effects upon plantlet response to transplanting: 1) the use of osmotica in rooting medium such as polyethylene glycol (PEG) or salts (NaCl + CaCl2) at different concentrations and durations, and 2) the use of semipermeable closures (SPC) which improves gas permeability of culture vessels. Concentrations of 1.0%, 2.5%, 5.0%, 10.0% and 15.0% PEG reduced water loss of detached leaves but affected growth adversely especially when above [5%] and 6-day-duration. PEG treatments induced epicuticular wax (EW) build-up and were related to reduced rates of water loss. Salt treatments reduced water loss at 1.5 and 2.0% over control but caused undesirable growth characteristics. Leaf diffusive resistance of PEG-treated plants was reduced prior to transplanting and remained lower than that of control. Stomates of IVPs had slower response to reduced humidity and darkness than greenhouse and PEG-treated plants. The SPC-treated cultures had 2.5 times more evapotranspiration, 2% less relative humidity (RH) and 3 times less medium and plantlet leaf water potential than B-cap-treated cultures. SPC treatments increased plantlet EW by 35%, reduced water loss by 60%, increased plantlet dry weights, reduced wilting injury and increased initial relative growth rates as compared to B-cap treatments. Photosynthetic rates of in vitro plantlets were reduced at RH lower than 80- 90%. SPC improved photosynthetic rates under desiccating conditions by reducing initial conductance and transpiration. Photosynthetic rates of IVPs from both closure treatments were comparable to those of greenhouse plants at high humidity. Stomates of plantlets from both treatments did not respond to [CO2] and darkness compared to stomatal responses of greenhouse plants. Chlorophyll content was increased in the SPC over B-cap plantlets. Better gas exchange of SPC was observed as indicated by CO2 accumulation. Although both osmotica and closure treatments reduced moisture loss in detached leaves of IVPs to levels comparable to those of greenhouse plants, the moisture loss curves deviated from normal bi-phasal shape indicating lack of normal stomatal functioning.
Open Access
Propagation, pollen storage, and in vitro lemmatoxin production of endod (Phytolacca dodecandra L.)
(Colorado State University. Libraries, 1991) Demeke, Tigst, author; Hughes, Harrison G., advisor; Ross, Cleon W., committee member; Lee, Chi Won, committee member; Towill, Leigh E., committee member
Protocols for macropropagation and micropropagation of endod are described. High levels on IBA (19.7 and 39.4 mM) resulted in the greatest number of roots of semi-hardwood cuttings. Combinations of IBA and NAA did not improve rooting. Cuttings taken from the apical and medial regions of stem branches rooted faster than the basal ones, but there was no difference in rooting percentage at six weeks. Scarification of seeds resulted in faster germination. Over 71% seed germination was obtained under aseptic condition in the control, gibberellic acid treated and scarified seeds. MS medium containing 0.44 μM BA induced 3.1 shoots per explant in vitro using shoot tips. Nodal explants produced up to 4.7 shoots per explant. Vertical and diagonal placement of nodal explants resulted in greater shoot proliferation than horizontal placement in strains 3 and 17. IBA at 0.49 μM induced 90% rooting of in vitro shoots with low callus production. Successful rooting was obtained at 1/2 and 1/4 MS basal salt concentrations with 0.49 μM IBA. Preculturing shoot tips in an MS medium containing IBA and transferring to medium without hormones did not reduce callus production at the explant base. Nodal explants gave 80 - 85% rooting with a low frequency of callus formation (7.5%). In vitro rooted plantlets grew normally and flowered in a greenhouse. Over 70% pollen germination was achieved on a medium containing 10% sucrose and 161.8 μM H3B03. Pollen stored at 1±1°C and -175°C maintained viability for six months, whereas pollen stored at 24±2°C lost viability within one month. Pollen stored at -175°C for three months when used in pollination set normal fruits and seeds. The greatest amount of callus was produced from shoot tip explants using IBA and 2iP. Flower bud and pericarp explants produced the least amount of callus. A hemolysis assay was found to be effective in analyzing saponin content of samples. No hemolytic activity was evident in the callus samples tested. Extracts from callus samples tested negatively for molluscicidal potency. Highest hemolytic activity of plant samples was recorded in the fully · enlarged green berry stage. Seeds were found to be essential for hemolytic activity.
Open Access
Rapid micropropagation of cocoyam (Xanthosoma sagittifolium) Schott
(Colorado State University. Libraries, 1993) Sama, Anne E., author; Hughes, Harrison G., advisor; Johnson, Duane, committee member; Towill, Leigh, committee member
Cocoyam (Xanthosoma sagittifolium Schott) was rapidly micropropagated through early subcultures at four and six week intervals and division of the microshoots. Shoot tips of approximately 3-5mm in size were used in the initiation of cultures on a modified B5 basal salt medium for six weeks. Cultures were subsequently micro propagated successfully through several procedures, which included the use of a range of growth regulator levels. Levels of 0.05μM NAA with 5, 10 and 20μM BAP as well as 1, 2 and 4μM TDZ singly or in combinations at the initiation and multiplication stages, as well as on agitated and stationary liquid media. Cultures initiation on solid media or supported on filter paper bridges was unsuccessful. However, solid media were beneficial in shoot multiplication, elongation and rooting stages. The level of 2μM TDZ alone and in combination with 20μM BAP significantly enhanced shoot proliferation, producing an average of 30 microshoots/culture, but repressed root formation. However, root initiation and development was possible in media containing only BAP at all levels tested. Shoot proliferation, elongation and rooting continued on media devoid of plant growth regulators and was independent of the culture vessel employed. A 100% survival of plantlets transferred into the greenhouse was achieved, irrespective of the method of acclimatization and size of the plantlet. Plantlets acclimatized in the humidity tent were less stressed, produced more and shed fewer leaves after two weeks from acclimatization. Differences in level of leaf injury were evident with control plants transferred directly to the open bench, which sustained the highest injury. The high level of survival of plantlets upon transfer to the greenhouse was attributed, in part, to the reduced numbers of stomates found on both abaxial and adaxial leaf surfaces of in vitro cultured plantlets, the high wax content and high rate of rooting.