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Tissue culture establishment and in virto studies of the creeping red fescue-didymella festucae interaction Suprayogi 1994

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TISSUE CULTURE ESTABLISHMENT AND IN CREEPING RED YESCUE-DIDYMELLA  VITRO  FESTUCAE  STUDIES OF THE INTERACTION  by SUPRAYOGI B.Sc, Universitas Jenderal Soedirman, Indonesia, 1986 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE in  THE FACULTY OF GRADUATE STUDIES (Department of Plant Science)  We accept this thesis as corifirrriing to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA November, 1994 © Suprayogi, 1994  In  presenting this  degree  thesis  in partial  fulfilment  of the requirements  at the University of British Columbia, I agree that the Library shall make it  freely available for reference and study. I further copying  for an advanced  agree that permission for extensive  of this thesis for scholarly purposes may be granted  department  or  by  his or  her  representatives.  It  is  by the head of my  understood  that  copying or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department  of  PUnl  £&te4U&i  <  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  &U*tbtr  ?$- Jqy • ,c  t  ABSTRACT  The use of tissue culture to study the underlying mechanism of creeping red fescue-Didymella festucae interaction was investigated using a cell culture-fungal  filtrate system. The study included optimization of protocols for tissue culture establishment and maintenance in creeping red fescue, and examination of the response of fescue cell cultures to D. festucae culturefiltratechallenge. Studies on tissue culture of creeping red fescue showed that not all cultivar and genotype sources were effective to be used to initiate cell culture materials for the study of creeping red fescue-D. festucae interaction. Among the initiated cell cultures, only the non-regenerable cell cultures of 'Boreal' and 'Cindy' could be used effectively for the above purpose. Growth of fescue cell suspension cultures was significantly suppressed by fungal filtrate challenge. Suppression was higher as concentrations of fungal filtrates were increased. The putative phytotoxic effect of culture filtrate was found to be heat sensitive and unlikely a cell wall component. It has not been determined whether this effect reflects the in planta disease interaction, or whether it is a phenomenon specific to the culture system environment. No significant difference (P < 0.05) was observed between phenylalanine ammonia-lyase (PAL) activity in fungal filtrate-challenged versus control fescue cell cultures. The time course of PAL activity following challenge with fungal filtrates varied extensivelyfromone sample period to the next and could not be used as a marker of the biochemical response of fescue cell cultures.  ii  TABLE OF CONTENTS page: ABSTRACT  ii  TABLE OF CONTENTS  iii  LIST OF TABLES  iv  LIST OF FIGURES  v  ACKNOWLEDGEMENT  vii  DEDICATION  viii  INTRODUCTION  1  LITERATURE REVIEW  3  CHAPTER I: EFFECTS OF LIGHT AND DARK INCUBATIONS ON CALLUS INDUCTION AND DEVELOPMENT IN CREEPING RED FESCUE Introduction 13 Materials and Methods 15 Results 17 Discussion 31 CHAPTER II: STUDIES OF THE CREEPING RED INTERACTION Introduction Materials and Methods Results •. Discussion  IN VITRO FESTUCAE  VESCUE-DIDYMELLA  35 40 44 55  GENERAL DISCUSSION  60  CONCLUSIONS  63  LITERATURE CITED  65  APPENDICES  70  iii  LEST OF TABLES page: Table 1. Analysis of variance, orthogonal contrast and Tukey's Studentized Range (Honestly Significant Difference) Test of data of frequency of callus induction in cultivars 'Boreal', 'Cindy' and 'Barcrown' (data were analyzed using General Linear Model of SAS) Table 2. Description of the type of callus tissue developed in light incubation  19 23  Table 3. Percentage of each callus type initiated in each cultivar in the light .... 26 Table 4. Percentage of each callus type initiated in each cultivar in the dark Table 5. Percentage of shoot-producing calli (type 3 calli) in cultivars of creeping red fescue prior to transfer onto plant regeneration (shooting) media (observation was done for 4 months)  26  27  Table 6. Analysis of variance, orthogonal contrasts and Tukey's Studentized Range (HSD) Test of data of callus weight increase at the end of second three-week incubation (data were analyzed using General Linear model of SAS)  29  Table 7. Analysis of variance, orthogonal contrast and Tukey's Studentized Range (HSD) Test of data of callus weight increase at the end of third three-week incubation (data were analyzed using General Linear model of SAS)  30  Table 8. Analysis of variance, orthogonal contrast and Tukey's Studentized Range (HSD) Test of data of cell fresh weight of cultures grown in media containing different treatments of fungal filtrates of D. festucae isolate 22170  48  Table 9. Analysis of variance, orthogonal contrast and Tukey's Studentized Range (HSD) Test of data of cell fresh weight of fescue cell cultures grown in media containing cell-free filtrate, crude homogenate of D. festucae isolate 22170 or controls  51  iv  LIST OF FIGURES page: Figure 1. Three different type of calli of creeping red fescue cultivar 'Boreal' initiatedfromcaryopses on full strength MS solid basal media and maintained for 3 months under continuous fluorescent lighting  20  Figure 2. Three different type of calli of creeping red fescue cultivar 'Cindy' initiatedfromcaryopses on full strength MS solid basal media and maintained for 3 months under continuousfluorescentlighting  21  Figure 3. Three different type of calli of creeping red fescue cultivar 'Barcrown' initiatedfromcaryopses on full strength MS solid basal media and maintained for 3 months under continuousfluorescentlighting  22  Figure 4. Individual plantlets of creeping red fescue cultivars 'Boreal', 'Cindy' and 'Barcrown' regeneratedfromtype 3 calli  24  Figure 5. Mycelial growth of D. festucae initiatedfrom6 agar plugs (0.5 cm diameter) of mycelial colony in a 100 ml potato dextrose broth medium and incubated on a gyratory shaker at 100 rpm at room temperature of 25 ± 3°C and normal room lighting (5.7 umol s" m"). • =freshweight, = dry weight  45  Figure 6. Growth of fescue cell cultures in half strength MS basal media added with hot (H) or cold (C) sterilized culture filtrate of different isolates of D. festucae. The treatments of basal media + none, basal media + basal media, basal media + water, and basal media + PDB were used were used as controls. Each point represents the mean of 6 observations  47  1  2  A  Figure 7. Growth of fescue cell cultures in half strength MS basal media added with cell-free filtrate, crude homogenate of D. festucae. The treatment of basal media + PDB and basal media + water were used as controls. Each point represents the mean of 10 observations Figure 8. Growth suppression in fescue cell cultures in response to different concentrations of crude homogenate of 3 isolates of D. festucae. Each point represents the mean of 5 observations done at the end of an 8-day-incubation period. = isolate no. 22168, • = isolate no. 22169, and • = isolate no. 22170  50  A  v  53  Figure 9. Effect of D. festucae crude homogenate on the induction of PAL activity in fescue cell cultures. Each point represents the mean of 5 observations. • = dH20 treated cell cultures, = fungal filtrate treated cell cultures. The straight lines represent the regression analysis for the control (dH20) ( ) and the fungal filtrate treatments (—). Specific PAL activity was expressed in pKatal per jig extracted protein in each sample. The concentration of the total extracted protein in each sample was deterrnined using Bradford method A  vi  54  ACKNOWLEDGEMENT aiiwaiflnii  Praise be to Allah, Most Gracious, and Most Merciful. The author is grateful to Dr. F. B. Holl as the research supervisor, for his patience, encouragement and assistance during the preparation of this thesis. His guidance about how science works is sincerely acknowledged. The author also thanks the supervisory committee, Dr. R. J. Copeman and Dr. J. E. Carlson; the examiners, Dr. J.W. Kronstad and Dr. C. P. Chanway, for their constructive comments and criticisms. Sincere thanks are extended to The Higher Education Development Project (HEDP) II - World Bank XXI, The Department of Education and Culture, Indonesia, for providing the initial scholarship, and to The Western Canada Turfgrass Association (WCTA), British Columbia, for generously providing supplemental financial support. Finally, thanks are due to the B. E. Ellis and F. B. Holl laboratory groups for sharing their knowledge and experience, Soenarto for his deft-hand with the SAS program, and The Department of Plant Science staff and graduate students for their kindness over the past three years.  vii  To my parents, and my family: Hardiyati, Lutfan and Luthfie, without whose enduring and unselfish support this would not have been undertaken  viii  INTRODUCTION  Creeping red fescue (Festuca rubra L. var.  rubra)  is one of the representatives  of turf grasses that is extensively planted for lawn and pasture purposes in North America (Aiken and Darbyshire, 1990). Its diverse uses for urban lawn, recreational turf and cometvation/reclamation applications have given it a significant role in the multi-million dollar turf grass industry. This includes seed production to supply market demand. Despite considerable research on red fescue as an important turf grass, less research has been conducted on seed production, and in particular, the occurrence of seed yield-reducing fungal disease. Seed production of creeping red fescue in the Peace River region of Alberta and British Columbia, may be significantly affected by the impact of diseases (Smith and Elliott, 1970). Stem eyespot, caused by Diaymella festucae (Weg.) Holm, is one of the fungal diseases that affects seed production. Production losses up to 50% are not uncommon in seed crops of creeping red fescue upon a disease outbreak. The disease is characterized by elongated eyespots on culms andflowerstalks, followed by dark green or brown stains (Martens et al., 1984). No disease controls have been effective to cope with fescue stem eyespot. As most damage is done on the culms andflowerstalks, application of fungicide may result in seed-yield reduction due toflowerdamage. While agronomic management gives only a temporary control, breeding for stem eyespot resistance in creeping red  1  2 fescue is hindered by the lack of resistant field-grown germ plasm. Therefore, establishment of a breeding program which incorporates new technologies to improve genetic resistance to stem eyespot disease would be a desirable long term solution. To facilitate such an approach, determination of basic physiological processes which underlie the creeping red fescue-D. festucae interaction would be very important to undertake. Unfortunately, determination of such physiological processes seems to be impractical unless plants are bearing culms. Tissue culture systems have been shown to be capable of providing a unique system for the study of the physiological processes of some plant pathogen interactions, as well as for genetic improvement of the related host plant. Therefore, it was of particular interest to determine if a tissue culture system was also amenable for the study of creeping red fescue-Z). festucae interaction.  LITERATURE REVIEW  1. Hie botany ofredfescue  Red fescue (Festuca rubra L.) is widely distributed throughout much of North America because of its broad climatic adaptation (Elliott and Bolton, 1970; Aiken and Darbyshire, 1990). Its dense, uniform growth, and Uirf-forming properties make it one of the finest lawn grasses, especially for drier and partly shaded sites (Aiken and Darbyshire, 1990; Dore and McNeill, 1980). Taxonomy of red fescue has been complicated by widespread selective breeding and the introduction of cultivars into Canadafromaround the world. The genus Festuca includes an estimated 450 species (Aiken and Darbyshire, 1990). The single species F. rubra is composed of a complex of native and introduced variants, the classification of which is not yet completely understood. Among commercial cultivars of red fescue, three groups have been recognized: octoploid creeping, hexaploid noncreeping, and hexaploid creeping red fescues; they are distinguishable by variation in their creeping habits. Creeping red fescue, Festuca rubra var. rubra, with 56 chromosomes, spreads by strong underground stems;  examples are cultivars 'Boreal' and 'Reptans'. Chewings fescue, Festuca rubra var. commutata Gaud., syn var. fallax Thuill., with 42 chromosomes, is tufted and does not  spread. The foliage of this sub-species is finer textured and the culms are much  3  4 shorter than that of creeping red fescue. A typical cultivar is 'Highlight'. The third group, also with 42 chromosomes, is intermediate in stature between chewings and creeping red fescues, and forms short rhizomes. This group is often referred to as slender creeping red fescue; an example is cultivar 'Dawson'. All red fescues have deep-feeding roots (Elliott and Bolton, 1970). Leaves are basal, shiny, folded, and bright green except for the reddish lower sheath. Culms are nearly leafless, shiny, and up to three feet tall for the strong creeping types. The seed head is a closed panicle with purplish-tinged spikelets. Seed hulls are awned and there are about 615,000 seeds per pound (Elliott and Bolton, 1970). Sixty seven species of pathogenic or decay fungi have been reported to associate with at least nine Festuca species in Canada (Conners, 1967; Ginns, 1986). Most of them attack pasture plants. rJuring the 1967 disease survey, Smith and his co-workers consistently isolated a stem eyespot-causing fungus, D. festucae, from lesioned plant materials (Smith et al., 1968). This seed-borne fungus was found associated with stems, sheaths, and inflorescences.  2. Stem eyespot disease in creepingredfescue  Stem eyespot has been reported as one of the fungal diseases affecting seed production in creeping red fescue (Smith and Elliott, 1970). The original description of the disease, 'leaf and pedicel blight', has been modified to 'stem eyespot' since  5 characteristic eyespot, stairring and extensive blackening of culms and inflorescences are the common symptoms (Smith and Shoemaker, 1974). The disease does not affect hay or pasture crops, but is severe on native fescues in the prairie and very destructive to commercial seed crops of creeping red fescue, particularly in wet years. It may cause seed losses of up to 50% in susceptible varieties (Martens et al., 1984). Disease severity seemed to be related to cropping intensity, which is higher in the more humid cleared parkland of the Peace River region. In a 1969 disease survey, stem eyespot disease was found on red fescue (F. rubra subsp. rubra), chewings fescue (F. rubra  subsp. commutata Gaud.), tall fescue (F. arundinacea Schreb.), and meadow  fescue (F. pratensis Huds.) at Beaverlodge (Smith and Elliott, 1970). The cultivar 'Olds' was considered both as the initial source of inoculum and the carrier of susceptibility genes, since many commercial creeping red fescue cultivars in North America were derivedfromthis genetic material. The disease may also have spread to introduced fescuesfromnative species in which the pathogen appears endemic.  1. Didymella festucae, the stem eyespot-causing fungus Didymella festucae (Weg.)  Holm, is the fungal pathogen causing stem eyespot  in seed crops of red fescue (Smith and Shoemaker, 1974). Its imperfect state, Phleospora idahoensis  (Sprague), has also been isolatedfromdiseased plant materials  (Smith et al., 1968). Despite an established taxonomic relationship between the perfect state, D. festucae, and its imperfect state, P.  idahoensis,  the latter name is still  ill-defined (Smith and Shoemaker, 1974). More recent papers refer to the stem  eyespot fungus as D. festucae (Smith and Shoemaker, 1974; Martens et al., 1984; Davidson and Klein-Gebbinck, 1991). In this thesis, therefore, unless it is used for specific taxonomic description, the fungus will be referred to as D. festucae. Ascocarps (pseudothecia) of the Didymella state in North American collections are sub-epidermal. Ascocarps, which ripen in spring and summer, are arranged in rows to the side or between the vascular strands of the stems or sheaths on blackened areas (gray-black when dry). All attempts to induce ascocarp production in culture have failed (Smith and Shoemaker, 1974). Typical pycnidia and spores of P. idahoensis developed in cultures derivedfromsingle ascospores of D. festucae isolated  from F. idahoensis, F. occidentalis, and F. rubra (Smith and Shoemaker, 1974). The pycnidia of P. idahoensis are very inconspicuous. On over-wintered stems the pycnidia are usually more obvious, especially when moistened with water, appearing as rows of dark blisters on the blackened stems (Smith et al., 1968).  2. Host range and Pathogenicity  In western North America, the perfect state, D. festucae, has been found on F. idahoensis Elmer, F. scabrella Torr., F. rubra L., F. ovina L. var. saximontana (Rydb.)  Gleason, F. occidentalis Hook., F. subuliflora Hook., and F. altcdca Trin. Conidia of the imperfect Phleospora state were recoveredfromall except the last two species. Typical stem eyespot symptoms have also been observed on F. pratensis Huds. and F. arundinacea Schreb (Smith and Elliott, 1970). Stem lesions also occurred on Phleum pratense L., Bromus inermis Leyss, and Agropyron spp. Isolatesfromthe latter two  7 grasses were typical of P.  idahoensis  but did not sporulate (Smith and Elliott, 1970).  In moist chambers, tillers of F. occidentalis, altcdca,  and F.  ovina developed  F. rubra, F. scabrella, F. idahoensis, F.  lesions when inoculated in leaf axils with a drop of  conidial suspensionfromcultures of two monoascoporic isolatesfromF. rubra (Smith and Shoemaker, 1974). Confirmation of the natural host range using pathogenicity tests with isolates from different hosts (cross-infection) confirmed that D. festucae has a wide, natural host range (Smith and Shoemaker, 1974). Cross-infection tests gave no indication of isolate specificity, but there was evidence for differences in virulence in different isolatesfromthe same host, e.g., F. rubra. There was also some evidence of resistance to the pathogen in a small number of the F. rubra and F. pratensis clones, but there was no indication of immunity. The resistance observed in this test, however, has not likely been recovered in cultivars grown under field conditions. In general, the cultivated cultivars of creeping red fescue are susceptible to stem eyespot.  3. Disease symptoms D. festucae infection  is easily recognizable by the characteristic dark spots on  sheaths and culms of affected grasses. The finding of blackened or stained overwintered culms provides a useful first step in the field recognition of the disease, although other disease organisms may cause similar discoloration (Smith and Shoemaker, 1974). Although inflorescence may have lesions, the typical symptoms on all Festuca  8 spp., linear spotting and blackening, are usually found only on leaf sheaths and culms with the primary damage to the culms. Leaf blades are rarely infected. On flowering culms, symptoms vary from vague-brown or brown-purple spots, linear in outline, through sharp brown linear streaks to clear eyespot formation with dark purple-brown margins and white or gray-white centres. Lesions usually do not exceed 1 cm length but are occasionally confluent.  4. Disease distribution D. festucae  has been found on F.  idahoensis  to extend south from Washington  through Oregon to the California-Nevada border, north in Saskatchewan almost to the Precambrian Shield and east to western Manitoba. In British Columbia, the disease has been found generally on F. ovina The result of the 1972 crop survey showed that the disease was severe on F. rubra and extended from the main growing region in northern Alberta to the fringes of areas where the disease had previously not been reported. Typical lesions, but no spores, were also found on overwintered stems of the F. rubra  collected as far north as the Yukon (Smith and Shoemaker, 1974).  5. Disease control  Efforts have been made to counteract stem eyespot disease. Some effects of the disease appeared to have been minimized by the use of high initial seeding rates (6-10 kg/ha), and by the application of nitrogen (50-70 kg/ha) in late fall. Burning, as part of stand rejuvenation, has also given some disease control, but it is not always  feasible nor effective after harvest. Even though these operations tend to improve yieldsfromthe first crop after treatment, none of them results in much control of the disease (Martens et al., 1984). Little genetic resistance to stem eyespot of field-grown cultivars is known (Martens et al., 1984). An indication of resistance has been observed in a cross-infection test (Smith and Shoemaker, 1984). However, this result was unlikely reproducible under field condition since no field-grown cultivar of creeping red fescue has been reported to be resistant to stem eyespot.  3. Tissue culture and itsrationalefor the study of plant-fungus interaction  Tissue culture has conventionally been used for rapid, mass plant propagation, and as a potential source for production of secondary metabolites. Its application has been extended for breeding purposes to cell selection, somaclonal variation, protoplast fusion and genetic engineering. Historically, tissue culture has contributed to plant disease control through propagation of pathogen-free plants, or cell selection for disease resistance. More recently, the study of host-pathogen interactions, as well as other physiological and molecular aspects of plant pathology has been carried out via the use of callus, cell suspension or protoplast cultures with assorted plant pathogens (Earle, 1978; Helgeson, 1983). The main reasons for the use of tissue culture include the highly standardized culture conditions, the morphological and physiological homogeneity of cultured cells  10 and the possible use of highly efficient selection techniques (Buiatti et al., 1985). In vitrv applications  may also offer other advantages to the in planta system for host-  pathogen interaction studies through control of the growth environment via medium constituents and temperature (Helgeson, 1983). Individual cells can also be observed from the moment of fungal inoculation. These advantages can be combined with a potential for rapid and homogeneous application of inoculation or other treatments to a more uniform cell population under controlled and aseptic conditions. Filter-sterilized or autoclaved toxin solutions can easily be added to nutrient media, thus eliminating problems that might arise when a living microorganism is added to a plant tissue culture (Earle, 1978). Supporting the use of plant tissue culture as a tool for breeding resistance, Buiatti et al. (1985) observed a strict correlation between vitro  behavior of the Dianthus  caryophylliis-Fusarium oxysporwn  in vivo  and  in  interaction.  Similarly, Gray et al. (1987), found similar results between the response of soybean plants to Phialophora gregata, a brown stem spot-causing fungus, and soybean callus to the fungal filtrates. In spite of the advantages, cell culture systems may also have some limitations (Helgeson, 1983). A correlation between filtrates and  in vivo  in vitro  response to phytotoxins or fungal  behavior is a necessary condition for effective use of plant tissue  cultures as a tool for the study of host-pathogen interactions and as an aid in resistance breeding (Buiatti et al., 1985). However, the events that occur in intact plants simply do not occur in some tissue cultures, and events occurring in cultures may not reflect those that occur in plants (Helgeson, 1983). As the defensive reactions that can be  11 studied in vitro are dynamic reactions, static defensive barriers such as the cuticle or pre-existing inhibitors are less likely to be represented in culture. Furthermore, defense mechanisms that are dependent on intercellular communication may be more difficult or impossible to examine with culture systems. For example, Budde and Helgeson (1981), found that the accumulation of phytoalexin in tobacco cell culture did not correlate with resistance to Phytophthora parasitica var. nicotianae in intact plants. Vardi et  al.  (1986) observed that culture filtrate of Phytophthora citrophthora  cannot be used as a selection tool in vitro since it produced indole acetic acid in axenic cultures that even promoted a better growth of cell cultures of a susceptible citrus cultivar. The use of purified phytotoxins, however, is not necessarily reliable since not all phytotoxins are important in disease development (Earle, 1978), or they represent only a part of the disease complex (Jones, 1990).  4. Research objectives  The mechanism by which D. festucae injures creeping red fescue is not understood. Unfortunately, unless the plants are bearing culms and inflorescences, determination of the mechanism of interaction between creeping red fescue and D. festucae, and  that of its resistance/susceptibility in planta seems to be impractical.  Therefore, it is desirable to find an appropriate system that can mimic the creeping red fescue-D. festucae interaction in planta  12 In vitro culture of plant cells and tissues  was shown to be capable of  providing a unique system for studying the basic physiological processes of some plant-pathogen interactions, as well as for genetic improvement of the related host plant. As the long term objective of the present research project is breeding of creeping red fescue for stem eye-spot resistance using tissue culture techniques, it is important to determine whether or not the in planta creeping red fescue-D. festucae interaction can be simulated and studied in vitro using a cell culture system. This research was divided into two sub-projects. The first part was concerned with the optimization of a tissue culture system for creeping red fescue. The objectives were to examine the growth and development of callus/cell cultures of creeping red fescue, and to determine the optimum conditions for culture maintenance. The second part focused on the study of axenic culture of D. festucae and of its effect on cell cultures of creeping red fescue. The objective was to examine the response of fescue cell cultures to culture filtrates of D. festucae. If consistent effects of culture filtrates of D. festucae can be demonstrated, many further studies on the creeping red fescue-Z). festucae in vitro interaction will be possible.  CHAPTER I EFFECTS OF LIGHT AND DARK INCUBATIONS ON CALLUS INDUCTION AND DEVELOPMENT IN CREEPING RED FESCUE  1. Introduction  Research on tissue culture of turf grasses has not been as extensive or progressive as for other plant species. However, there are a number of species that have been subjected to sufficient study so that tissue culture technology can now be applied to improve these species (Krans, 1985). Krans (1981) screened 16 turf grass species for callus initiation and plant regeneration. This research has led to more detailed studies on hormonal and nutritional effects on callus induction and plant regeneration for creeping bentgrass  (Agrostis palustris  Huds.) (Krans  et al., 1982),  Kentucky bluegrass (Poapratensis L.) (Manton et al., 1981) and perennial ryegrass (Lolium perenne  L.) (Torello and Symington, 1984). Moreover, Ha et al. (1992) have  developed transgenic tall fescues {Festuca arundinacea Schreb.)fromprotoplasts via electroporation. Research on tissue culture of creeping red fescue was first undertaken by Torello and his co-workers (Torello et al., 1984). In their initial study, optimum hormonal and nutritional requirement for callus induction and plant regeneration were described. From two different cultivars of creeping red fescue, it was shown that at least two morphologically distinct types of callus tissue, embryogenic and 13  14 nonembryogenic calli, were produced. Increasedfrequencyand duration of plant regeneration were attributed to embryogenic callus. This research was followed by work focused on the culture behavior of creeping red fescue (Torello et al., 1984; Zaghmout and Torello, 1988, 1989, 1990 and 1992). Despite satisfactory resultsfromprevious research on creeping red fescue, it seems likely that not all aspects of in vitro culture have been addressed thoroughly. Information about the rationale for using dark incubation for callus induction by Torello and his co-workers is lacking. Krans (1981) observed inconsistent effects of light and dark incubations on callus induction and plant regeneration of turf grass species. His results showed that the observed effects were likely dependent on the cultivars tested. There is always a possibility that different cultivars have different patterns of callus/cell culture development in response to light and dark. Because of these uncertainties, and the different genetic material used in this thesis research, it was necessary to determine the effects of light and dark incubations on the induction and development of callus in creeping red fescue. The results of these experiments were used to determine the culture protocol for subsequent analyses.  15 2. Materials and Methods  Medium All experiments were conducted on Murashige and Skoog (MS) media (Murashige and Skoog, 1962). Full strength MS basal media were used for callus induction. Cell suspension cultures were maintained in agar-free, one-half-strength MS media. Regeneration media were growth regulator-free, one-half-strength MS solid media; while rooting media were one half strength MS solid media containing indole butyric acid (IBA) ( 0.5 mg per liter). Regardless of the strength of the basal medium, 30 g sucrose was added to every 1000 ml MS basal salt solution. To make a solid medium, 12 g Bacto Difco agar was added to 1000 ml MS basal salt solution. The pH of all media was adjusted to 5.80 ± 0.2. Vitamins and growth regulators were added to the medium before autoclaving. The ingredients, composition and strength of the media used are presented in Appendices 1 and 2. Turf grass cultivar The turf grass cultivars tested in this study included 'Boreal', 'Cindy' and 'Barcrown'. Certified seeds of the cultivars were provided by Richardson Seed Company Limited of Burnaby, British Columbia. Callus induction Torello's protocol (Torello et al., 1984) was adopted for culture establishment of creeping red fescue. Mature caryopses were used as explant sources for callus induction. Caryopses were dehusked in 50% (v/v) H S0 for one minute, followed by 2  4  16 rinsing with sterile distilled water. The caryopses were then surface sterilized in 1% (v/v) sodium hypochlorite for 30 minutes followed by three rinses in sterile distilled water. Ten caryopses were placed on a full strength solid MS medium in a petri dish. For each cultivar, 40 petri dishes were prepared. Twenty petri dishes were incubated in the dark, while the remaining group was incubated under continuous fluorescent light (7.8 umol s" m"). The initiated calli were transferred to new media at the end of 1  2  a six week-incubation period by removing callusfromshoot, root and/or caryopsis tissue. Callus inductionfrequencies(as %) were determined, and callus weight increases were measured in two subsequent incubation periods following the first subculture. Callus inductionfrequencieswere compared using ANOVA and orthogonal contrasts using the general linear model of SAS. Descriptive observations and classifications of callus type were also made for each of the three cultivars. Culture maintenance Calli chosen for subsequent experiments were maintained by subculturing them onto afreshfull strength solid MS medium at 4- to 6-week intervals. At the fourth subculture, each callus derivedfroma single caryopsis was transferred individually onto a separate petri dish. Cell suspension cultures were prepared by placing approximately 3 g of callus into 30 ml agar-free, one-half-strength MS medium. The cell suspension cultures were incubated under continuous lighting (7.8 umol s" m") on 1  a gyratory shaker at 100 rpm, and subcultured at four week intervals.  2  17 3. Results  In general, the published protocols for callus induction and maintenance worked effectively for all the three cultivars tested. Slight modifications were made through preliminary trials to optimize the culture system. For example, 1.2% (w/v) agar (Bacto Difco) was used for the solid medium. The sucrose concentration of 20 g per 1000 ml MS basal medium did not promote good cell culture growth. A better growth was observed when the sucrose concentration was increased to 30 g per 1000 ml MS basal medium. The subculture period was also changed from 8-week to 4week intervals. No significant difference (P < 0.05) was observed between the effects of light and dark incubations on callus induction in any cultivars tested (Table 1). Differences infrequencyof callus induction were observed among different cultivars. A significant difference was also observed among replications. Callus induction in cultivar 'Boreal' was significantly higher than those in cultivar 'Cindy' and 'Barcrown' . No significant difference was observed between callus induction in the latter two cultivars. The averagefrequenciesof callus induction in cultivar 'Boreal' and 'Cindy'/'Barcrown' were 81.75 ± 1.25% and 62.5 ± 4.5% respectively. It was anticipated that once callus had been initiated it could be maintained as callus in the absence of any specific treatment with growth regulators or otherwise modified media. In this experiment, however, differences in callus development were observed among and within cultivars before transfer to a modified medium. Three  18 types of callus tissues were observed in both light and dark incubations. Photographs and descriptions of each type of callus tissue for each cultivar are presented in Figures 1, 2, 3, and Table 2 respectively. The pattern of development of dark-incubated calli was similar to that of lightincubated calli, except that the color of calli and any developing shoots was paler. The grouping criteria for dark-incubated calli, therefore, were similar to those for light-incubated calli. An attempt to regenerate plantlets was also madefromcallus by placing 14 to 16 week old callusfromeach caryopsis (genotype) onto a shooting medium for a month, followed by transfer of the shoot and callusfragmentsonto a rooting medium in a culture tube. This attempt was initially intended to determine plantlet regeneration capacity in each callus type in each cultivar. This objective was somewhat confounded by the formation of shoots and roots in some calli before subculture onto shooting or rooting medium. Specific determination of shoot number per callus prior to and after transfer to modified media could not be recorded since the calli which produced shoots did so in an extremely prolific manner. Shoots developed from almost all of the outer cells of calli which resulted in the formation of denseshooted calli. Encountering a similar problem, Torello et al. (1984) used the term 'an extremely prolific shooting' to describe such shootings. Therefore, only qualitative observations were made to deteirriine whether or not plantlets could be regenerated from each type of callus to support the descriptive differentiation of callus development.  19  Table 1. Analysis of variance, orthogonal contrasts and Tukey's Studentized Range (Honestly Significant Difference) Test of data offrequencyof callus induction in cultivar 'Boreal', 'Cindy' and 'Barcrown' (data were analyzed using General Linear Model of SAS) F Value  Pr>F  Type III SS  Mean Square  369.5917 67.7583 107.9416  3.5662 21.5883  1.75* 10.58**  0.0413 0.0001  3.0083  3.0083  1.47  0.2277  Within light incubations: cl vs c2-3 1 46.8750 c2 vs c3 1 3.0250  46.8750 3.0250  22.97** 1.48  0.0001 0.2265  Within dark incubations: cl vs c2-3 1 52.0083 c2 vs c3 1 3.0250  52.0083 3.0250  25.48** 1.48  0.0001 0.2265  Source  df  Total Replications Treatments  119 19 5  light incubation vs dark incubation 1  Error  95  193.8917  2.0410  Legend: cl c2 c3 *, **  = frequency of callus induction in Boreal = frequency of callus induction in Cindy = frequency of callus induction in Barcrown = significant at P < 0.05 and P < 0.01, respectively  Tukey Grouping  Mean  N  Treatment  A A  83% 80.5% 67% 63.5% 61.5% 58%  20 20 20 20 20 20  1 =light x Boreal 4 = dark x Boreal 3 = light x Barcrown 6 = dark x Barcrown 2 = light x Cindy 5 =dark x Cindy  B B B B  N = number of replicates Means with the same letter are not significantly different  \  23 Table 2. Description of the types of callus tissue developed in light incubation Type  1  Boreal  Cindy  - friable and soft - fast growing - whitish - good for callus culture - good for cell culture - no shooting - no plant regeneration '  -  friable and soft fast growing whitish good for callus culture good for cell culture no shooting ' no plant regeneration '  -  friable and soft fast growing whitish greenish fairly good for callus culture not good for cell culture some shootings ' good plant regeneration '  -  compact and soft slow growing whitish brownish not good for callus cult. not good for cell culture no shooting no plant regeneration '  -  compact and soft slow growing whitish brownish not good for callus cult. not good for cell culture no shooting ' no plant regeneration '  -  compact and fairly soft slow growing whitish greenish not good for callus cult. not good for cell culture no shooting ' poor plant regeneration '  -  friable and soft fast growing whitish greenish good for callus culture fairly good for cell cult. produce poor shooting ' good plant regeneration '  - friable and soft - fast growing - whitish greenish - good for callus culture - fairly good for cell culture - produce poor shooting ' - good plant regeneration '  0  2  2  3  '' 2)  Barcrown  0  2  1  2  1  2  1  2  1  2  1  2  1  2  - friable and soft - fast growing - whitish greenish - poor callus culture - not good for cell culture - produce vigorous shooting ' - very good plant regeneration 1  : prior to transfer to shooting media : after transfer to shooting media  The above descriptions were based on observationsfromthe first subculture up to the time of the plant regeneration trial (see Figure 4. for regenerated plantlets) and cell culture maintenance to allow a reliable judgment of callus development. It should be noted that due to differences in the pattern of callus development, the descriptions used for each type of callus tissue may be different within a similar type of callus among cultivars.  24  25 Observationfromthe first callus-subculture up to the plant regeneration trial and cell culture maintenance showed that type 1 calli of 'Boreal' and 'Cindy' were very good sources for callus and cell cultures, but type 1 calli of 'Barcrown' was not. The callus and cell suspension cultures of type 1 calli of 'Barcrown' were found to undergo tissue differentiation before transfer to modified media. Attempts' to regenerate plantletsfromtype 1 calli of cultivars 'Boreal' and 'Cindy' were not successful. Type 2 calli of all cultivars tested could not be used as sources for callus and cell suspension cultures, nor for plant regeneration. Type 3 callus of 'Barcrown' was found to continue producing shoots until no callus tissue remained. This callus also represented a poor source for callus and cell culture development. Type 3 calli of 'Boreal' and 'Cindy' could be used as sources for callus and cell suspension cultures, and for plant regeneration. All suspension cultures of this callus type, however, were found to undergo tissue differentiation. Tables 3 and 4 summarize the percentage (%) of each callus type in each cultivar in light and dark incubations. Percentage of each callus type was defined as the ratio of a given callus type to the total number of the initiated calli within a cultivar. Some criteria were used for grouping of the initiated calli. These criteria sometimes overlapped within the same callus type and/or within the same cultivar. To avoid confusion, only the criteria of the callus growth (fast/slow growing), the callus quality for cell culture (good/poor cell culture), and the callus regenerability for plantlets (no/poor/good plant regeneration) were used to count the percentage of each callus type within a cultivar. Since these data were not replicated, they were not  26 analyzed statistically. Table 3. Percentage of each callus type initiated in cultivars of creeping red fescue in the light Boreal Type  Cindy  Barcrown  total  (%)  total (%)  total  1 2 3  27 33 15  36 44 20  16 21 49 66 10 13  9 35 31  12 47 41  Total  75  100  75 100  75  100  (%)  Table 4. Percentage of each callus type initiated in cultivars of creeping red fescue in the dark Boreal Type  Cindy  Barcrown total  (%)  16 32 30 60 4 8 .  8 30 12  16 60 24  50 100  50  100  total  (%)  total (%)  1 2 3  25 23 2  50 46 4  Total  50  100  In Tables 2, it can be seen that calli of cultivars 'Boreal' and 'Cindy' tended to have a similar pattern of development - prolific growth but very poor shoot development. Calli of cultivar 'Barcrown' tended to be much more shoot-prolific than those of cultivar 'Boreal' and 'Cindy'. In cultivars 'Boreal' and 'Cindy', the percentages  27 of type 1 calli were higher than the percentages of type 3 calli. In cultivar 'Barcrown', the trend was reversed (Tables 3 and 4). In cultivars 'Boreal' and 'Cindy', the percentage of type 3 calli (Table 3 and 4) was similar to that of shoot-producing calli (Table 5). These percentages seemed to indicate total regeneration capacity of the calli. In cultivar 'Barcrown', however, the percentage of type 3 calli (Table 3 and 4) did not reflect total regeneration capacity (shoot-producing calli) (Table 5) since some of the type 1 calli of this cultivar also produced shoots before and after transfer to a modified medium. The percentage of shoot-producing calli (48% and 30%) (Table 5) in cultivar 'Barcrown' included calli of both type 1 and 3 (Table 3 and 4).  Table 5. Percentage of shoot-producing calli (type 3 calli) in cultivars of creeping red fescue prior to transfer onto plant regeneration (shooting) media (observation was done for 4 months) Cultivar  Light incubation  Boreal Cindy Barcrown  20% 13.33% 48%  Dark incubation 4% 8% 30%  Observation was also made at two subsequent culture periods to determine any further effects of light and dark incubations on callus growth. The first evaluation was made at the end of the second three-week culture incubation, followed by an additional examination at the end of the third three-week incubation. Callus growth was  28 measured as callus weight increase over the specified culture period. Orthogonal contrast analysis of data for the callus weight increase shows that there was a highly significant difference in callus growth between light and dark incubations (Table 6 and 7). However, the effects of light and dark incubations on callus growth in the second and the third three-week incubation were inconsistent. In the first incubation period, light incubation appeared to produce better callus growth; while in the second incubation period, the trend was reversed. There was no significant difference in callus growth among the three cultivars in both culture periods.  29 Table 6. Analysis of variance, orthogonal contrasts and Tukey's Studentized Range (HSD) Test of data of callus weight increase at the end of the second three -week incubation (data were analyzed using General Linear Model of SAS) Source  df  Total 89 Replications 14 Treatments 5  Type III SS Mean Square F Value  Pr > F  0.2651 0.0550 0.0479  0.0039 0.0096  1.69 4.13**  0.0766 0.0024  Light incubation vs dark incubation 1 0.0416  0.0416  17.94**  0.0001  Within light incubations: cl vs c2-3 1 0.0080 c2 vs c3 1 0.0018  0.0009 0.0018  0.37 0.77  0.5463 0.3834  Within dark incubations: cl vs c2-3 1 0.0001 c2 vs c3 1 0.0036  0.0001 0.0036  0.04 1.55  0.8515 0.2169  Error  0.0023  70  0.1623  Legend: HSD cl c2 c3 *, **  = Honestly Significant Difference = fresh weight increase in calli of'Boreal' = fresh weight increase in calli of 'Cindy' = fresh weight increase in calli of 'Barcrown' = significant at P < 0.05 and P < 0.01, respectively  Tukey Grouping  B B B B  A A A A A  Mean  N  Treatment  0.1374 0.1235 0.1155 0.0886 0.0871 0.0717  15 15 15 15 15 15  5 =dark x Cindy 4 = dark x Boreal 6 = dark x Barcrown 1 =light x Boreal 2 = light x Cindy 3 = light x Barcrown  N = number of replicates Means with the same letter are not significantly different.  30 Table 7. Analysis of variance, orthogonal contrasts and Tukey's Studentized Range (HSD) Test of data of callus weight increase at the end of the third three -week incubation (data were analyzed using General Linear Model of SAS) Source  df  Total 89 Replications 14 Treatments 5  Type III SS Mean Square F Value 96.3685 0.2284 0.2121  light incubation vs dark incubation 1 0.0792  Pr > F  0.0163 0.0424  0.86 2.25  0.5989 0.0589  0.0792  4.19*  0.0443  r  Within light incubations: cl vs c2-3 1 0.0352 c2 vs c3 1 0.0034  0.0352 0.0034  1.87 0.18  0.1763 0.6736  Within dark incubations: cl vs c2-3 1 0.0330 c2 vs c3 1 0.0615  0.0330 0.0615  1.75 3.26  0.1906 0.0754  Error  0.0189  70  1.3209  Legend: HSD cl c2 c3 *, **  = Honestly Significant Difference = fresh weight increase in calli of'Boreal' = fresh weight increase in calli of 'Cindy = fresh weight increase in calli of 'Barcrown' = significant at P < 0.05 and P < 0.01, respectively  /  Tukey Grouping Mean B B B B B  A A A A A  0.2118 0.1630 0.1512 0.1418 0.1390 0.0485  N  Treatment  15 15 15 15 15 15  1 = light x Boreal 2 = light x Cindy 4 = dark x Boreal 3 = light x Barcrown 5 = dark x Cindy 6 = dark x Barcrown  N = number of replicates Means with the same letter are not significantly different.  31 4. Discussion In this experiment, both light and dark incubations did not have different effects on thefrequencyof callus induction in the three cultivars tested. Differences in thefrequencyof callus induction were observed among cultivars. Thefrequencyof callus induction in cultivar 'Boreal' was significantly higher than that in 'Cindy' and 'Barcrown', but no significant difference was observed between the latter two cultivars. Thus, callus inductionfrequenciesare likely dependent on the cultivar sources. This result is consistent with the observations of Krans (1981) and Torello et al. (1984). Krans (1981) observed that callus induction in 16 turf grass species was dependent on the cultivar source and the medium kinetin/2,4-D ratio, but was independent of light or dark incubation. Torello et al. (1984) found that within the same species of Festuca rubra,  cultivar source was one of the factors that affected callus induction and  development. The result of analysis of variance showed a significant difference in the frequency of callus induction among replicates. This result was not surprising since caryopses have been used to initiate the calli. Creeping red fescue is a crosspollinated crop, hence the caryopses used for callus induction were heterogeneous. This heterogeneity has resulted in the variablefrequencyof callus induction among replicates. Further observations of developing calli prior to transfer to suspension culture and/or plantlet regeneration media, confirmed the development of three types of callus tissuefromeach cultivar. Torello et al. (1984) observed two types of callus in  32 creeping red fescue which he defined as 'embryogenic' and 'nonembryogenic' calli. Most cereal and grass cultures have also been shown to produce at least two morphologically distinct types of callus tissue, embryogenic and nonembryogenic (Krans et al., 1982; Manton et al., 1981; Wang and Vasil, 1982). In this experiment, however, no embryogenic calli have likely been initiated from caryopses of creeping red fescue even though Torello's protocols (Torello, 1984) have been used. Embryolike structures, an indication of embryogenesis, were not observed during the development of shoots from calli of all cultivars throughout this experiment. Visual observation showed that the shoots were developed directlyfromclusters of meristematic cells. These calli might have consisted of organogenic cells, since organogenesis in callus starts with the formation of cluster of meristematic cells (meristemoids) capable of responding to factors within the system to produce a primordium [Torrey (1966) in Dodd and Roberts, 1988]. Therefore, these^calli may be best defined as 'regenerative' calli. These calli included some of the type 1 calli of 'Barcrown' and most of the type 3 calli of all the three cultivars. Type 1 calli of 'Boreal' and 'Cindy' did not produce shoots and were recalcitrant for plant regeneration. These calli might be defined as 'non-regenerable' callus. These calli included the type 1 calli of'Boreal' and 'Cindy'. Type 2 calli did not grow and develop well. To avoid reporting misleading information, therefore, they were not grouped as non-regenerable nor as regenerative. The percentages of the types 1, 2 and 3 calli tended to varied among the cultivar tested, but not between light and dark incubations. In cultivars 'Boreal' and  33 'Cindy' the percentage of type 1 ('non-regenerable') calli was higher than that of type 3 ('regenerative') calli, while in cultivar 'Barcrown' the ratio was the reversed. Unless used for plantlet regeneration purposes, light was unlikely to be required for further development of calli after initiation in creeping red fescue. The development of callus after initiation was likely dependent on endogenous factors in the caryopses used as explants rather than on light or dark incubations. Torello et al. (1984) also observed that the development of calli after initiation in the species of F. rubra is dependent on the cultivar sources. Upon transfer into suspension cultures, type 1 calli of 'Boreal' and 'Cindy' formed dispersed single-celled suspension cultures and grew very well, while type 1 calli of 'Barcrown' formed clumped suspension cultures and grew poorly. None of type 2 and 3 calli of all cultivars tested could be used for cell suspension culture materials. Thus, only non-regenerable calli of creeping red fescue were suitable to be used for cell suspension culture materials. Observation of the effects of light and dark incubations on callus growth in two culture periods showed significant but inconsistent results. There was no difference in the callus growth of the three cultivars tested. The inconsistent effect of light and dark incubations in the two culture periods is more likely a consequence of physiological disturbances induced by subculture. Photosynthesis and other metabolisms of the light-incubated calli might be disturbed because of the subculturing. The metabolism of dark-incubated calli might also be affected due to exposure to light during subcultxiring. For callus cultures that are maintained through  34 routine subcultures, the effects of light and dark incubations on the callus growth can not likely be detemiined based on observations of only two culture periods. Based on these initial observations, it can be concluded that if direct plant propagation is the purpose of tissue culture work in creeping red fescue, 'Barcrown' is the best choice of the three cultivars tested. If the purpose of the tissue culture work is to provide a cell culture source for an festucae interaction,  in vitro study  of the creeping red fescue-D.  'Boreal' and 'Cindy' are likely to be more desirable source  materials. Effective use of these cultivars for eventual plant breeding will require additional research on plant regenerationfromtype 1 callus tissue of 'Boreal' and 'Cindy', and to produce an effective dispersed cell culturefromtype 1 callus tissue of 'Barcrown'. The effect of light and dark incubations on thefrequencyof callus induction was not significant. However, calli produced in the light tended to be greener and they responded more effectively to plant regeneration. Therefore, light incubation was the preferable condition for callus/cell culture maintenance and plantlet regeneration in creeping red fescue when regeneration was important. Based on the callus morphological description and the percentage of type 1 callus among the three cultivars tested, 'Boreal' was selected as the most appropriate culture material for additional experimentation.  CHAPTER II IN VITRO STUDIES OF THE CREEPING RED  FESCU E-DID  YMELIA  FESTUCAE INTERACTION  1. Introduction  Selection of genotypes resistant to D. festucae may be the most effective solution to coping with stem eyespot in creeping red fescue since agronomic management has not resulted in effective control. However, traditional breeding for resistance to stem eyespot in creeping red fescue suffersfromseveral drawbacks, such as the lack of genetic variability, the extensive time and space requirements for screening plant populations under selection, and a lack of reliable early screening methods. The solution to these problems may be the use of selection methods at the cellular level, screening techniques based on reliable biochemical markers for resistance, and genetic engineering for single genes affecting host-pathogen interactions (Buiatti and Ingram, 1991). To facilitate the use of such approaches detailed in vitro studies of the creeping red fescue-D. festucae interaction should first be undertaken. Many fungal and bacterial pathogens produce phytotoxins upon interaction with their hosts; these compounds injure the hosts even at low concentrations (Earle, 1978). Victorin (of Helminthosporium  victoriae),  HMT toxin (of if  maydis  race T)  and PM-toxin (of Phyllosticta maydis) are examples of such phytotoxins. Phytotoxins  35  36 have been isolatedfromdiseased plant materials, but they are usually obtained from filtered liquid cultures of the pathogen (Earle, 1978). Considerable evidence has suggested that purified phytotoxins or culture filtrates could be used as detenriinants of host resistance in vitro (Buiatti and Ingram, 1991). Experiments on the use of toxins/culture filtrates as markers for host resistance have shown a positive correlation between toxin/culture filtrate tolerance and resistance to pathogen (Buiatti et al., 1985; Gray et al., 1987). Studies of phytotoxin production by D. festucae are lacking. A representative of the same genus,  D. applanata  (Niessl.) Sacc, has been found to release a toxic  oligosaccharide on its host, the raspberry plant (Rubus  idaeus)  (Suchuring and  Salemink, 1972). A more recent study showed that the toxin was a glycopeptide (van Broekhoven et al., 1975). This fungus also produced a phytotoxin when cultured in a liquid medium; its culture filtrate was toxic to young raspberry sprouts placed in this liquid. No a priori assumptions may be made about the production, structure or mode of action of any putative toxin of D. festucae. However, in view of the observations for D.  applanata,  it may be not too speculative to hypothesize that D. festucae releases  a toxin to injure red fescue tissue. In axenic culture, D. festucae was observed to exude an age-related brown pigment into the medium when the fungus was grown at 18°C on potato dextrose agar (PDA) (Smith et al., 1968). Surface drops of exudate also appeared on the fungal colonies. In preliminary observations of D. festucae cultured in potato dextrose broth  37 (PDB), the pathogen also released a pigmented exudate into the medium. The possibility that D. festucae produced phytotoxin(s) in culture, and the observed correlation between phytotoxin/culture filtrate tolerance and host resistance to pathogens for some plant-pathogen interactions led to the development of in vitro studies of the creeping red fescue-Z). festucae interaction. Such studies were undertaken with the following objectives: 1. to examine the growth of D. festucae in liquid culture, 2. to determine the effect of hot-sterilized and cold-sterilized crude homogenates of D. festucae on the  growth of fescue cell suspension cultures,  3. to compare the effects of cell-free versus crude homogenate of D. festucae on the growth of fescue cell suspension cultures, 4. to determine the impact of different concentrations of crude homogenate of cultured D. festucae on the growth of fescue cell suspension cultures, and 5. to determine the time course effect of fungal crude homogenates on the induction of phenylalanine ammonia-lyase (PAL) (E.C. 4.3.1.5) activity in suspension cultures of creeping red fescue. The first experiment was carried out to deteimine the appropriate harvesting time of D. festucae cultures when grown in 100 ml of liquid PDB medium. It was assumed that the putative fungal toxin production would be maximized by the end of the exponential phase. For culture filtrate preparation, therefore, the fungal cultures were harvested at the time between the end of the exponential phase and the beginning of the stationary phase of the fungal growth.  38 Sterilization provides an extra-cellular filtrate uncontaminated by culturable fungal material (spores or mycelium) which may provide more direct evidence for a putative D. festucae toxin than a non-sterilized crude homogenate. However, autoclaving, a common method of sterilization, might result in destruction of toxin activity, or release of deleterious compounds which affect plant cell growth, but are unrelated to pathogenicity. The second experiment was designed to examine the effect of sterilization by hot (autoclaving) and cold (ultra filtration) treatments on cell growth. Some fungi injure their host plants by releasing their toxins to the host tissue (Suchuring and Salemink, 1972; Vardi et al., 1986; and Broekoven et al., 1986), while some of the others injure the hosts by disrupting the host tissue with their cell wall components (Doke and Tomiyama, 1980; Buiatti et  al.,  1985). If D. festucae injures  creeping red fescue by releasing an exudate, its cell-free filtrates might be used effectively to challenge the cell cultures. If its putative phytotoxic compound is a cell wall component (elicitor), homogenized fungal crude homogenates would be more effective. The third experiment was designed to determine which was more effective, the cell-free filtrate or crude homogenate, when used to challenge fescue cell cultures. The fourth experiment was designed to evaluate the effect of different concentrations of fungal crude homogenates on growth suppression of fescue cell suspension cultures. In addition to determining whether a dose effect might be observed, this experiment was used to define an appropriate treatment level of the fungal filtrate used in further experiments.  39 Many disease resistance-associated host molecules are products of secondary metabolism; phenylalariine ammonia-lyase (PAL) is believed to be the key enzyme in the formation of these compounds (Jones, 1990; Geoffrey, 1988). PAL catalyzes the first reaction in the biosynthesis of a wide variety of phenylpropanoid compounds including lignin, esters of hydroxycinnamic acids and flavonoids (Lamb et al., 1979). In some grasses and certain fungi,frtra-cinnamicacid and />coumaric acid, the precursors of a variety of phenylpropanoid compounds, are formedfromboth Lphenylalanine by PAL, andfromL-tyrosine by tyrosine ammonia-lyase (TAL)(Neisch, 1961; Jangaard, 1983; Hanson and Havir, 1981; Geoffrey, 1988). It has not been reported whether PAL and/or TAL function specifically in defense reactions against pathogens in creeping red fescue. Observation of the relative ratios of PAL and TAL in a number of grasses, has shown that in every case the specific activity of PAL was greater than that of TAL (Jangaard, 1973). PAL activity is more commonly used as a marker of a biochemical plant defense responses. In tobacco, for example, PAL activity was induced in resistant and susceptible callus tissues in response to both compatible and incompatible spores of Phytophthora parasitica var.  nicotianae  (Helgeson et al., 1978). Cahill and McComb (1992) also observed that PAL was induced in both resistant Eucalyptus  calophylla and  susceptible E.  marginata  root  segments infected with Phytophthora cinnamomi. Differences between susceptible and resistant tissue were reflected in the observation that PAL activity was significantly higher, and the induction was usually much earlier in resistant plant tissue. PAL activity may provide an easily measured marker to distinguish resistant and susceptible  40 deeping red fescue cultivars, even in the absence of a normal disease response. The fifth experiment was designed to determine if PAL activity and the time course of enzyme development could be used as a marker response of fescue cell cultures to treatment with crude homogenates of D. festucae.  2. Materials and Methods  Examination of the growth of D. festucae in PDB medium D. festucae isolate  no. 22170 (known to cause disease in F. rubra) obtained  from the American Type Culture Collection (ATCC) was used in this study. Difco potato dextrose broth (PDB) was used to prepare the liquid medium. Little or no success has been reported in attempts to induce sporulation of D. festucae in culture (Davidson and Kiein-Gebbinck, 1991); in the present studies, attempts to induce sporulation were also not successful. Fungal suspension cultures were initiated from agar plugs derivedfromthe colony border of fungus grown on potato dextrose agar (PDA). Six plugs (0.5 cm diameter) were added to each of the 250 ml flasks containing 100 ml sterile PDB. The cultures were incubated on a gyratory shaker at 100 rpm at room temperature of 25 ± 3°C and light intensity of 5.7 umol s" m". 1  2  Fungal growth was measured by deteirnination offreshand dry weights of mycelial mass. Measurements were madefromthe third day of inoculation for 8 days. Eight replicates were taken randomly for each measurement.  41 In vitro challenge experiments  Several preliminary experiments were undertaken to determine the impact of co-cultivation or challenge of the fescue cell cultures with Didymella festucae. These experiments indicated that growth suppression and browning (data not shown) of the cultures occurred in response to the addition of fungal extracts. These preliminary results were used to design the experiments described in the following sections.  Determination of the effects of sterilization method on the effect of fungal crude homogenates on the growth of fescue cell suspension cultures  Three isolates of D. festucaefromthe ATCC were used in this experiment: isolate no. 22168, isolate no. 22169 (known to cause disease in F. scabrella) and isolate no. 22170 (known to cause disease in F.  rubra).  Fungal crude homogenates  were preparedfrom8 day-old cultures. After harvesting, the cultures were blended at room temperature using an electric blender for three minutes and thenfilteredthrough three layers of Whatman filter paper No. 41. The filtrate was divided into two separate aliquots. One half of thefiltratewas autoclaved at 120°C for 20 minutes, while the other half was passed through a 0.22 um Millipore filter. Cell cultures of 'Boreal' fescue were prepared by placing approximately 3 grams (fresh weight) of cells into a 125 ml flask containing 24 ml agar-free, one-halfstrength MS basal medium. The cell cultures were allowed to grow on a gyratory shaker at 100 rpm under continuous room lighting (7.8 umol s" m") at 25°C for three 1  2  days prior to the addition of the fungal filtrates. On day four, 6 ml of fungal filtrate or control solution were added to each cell  42 culture to make up a 30 mlfinalvolume. Ten different treatments were used in this experiment. Hot and cold sterilized crude homogenates of isolates nos. 22168, 22169 and 22170 were used as main treatments. Four additional treatments, namely: no additional solution, sterile one half strength MS basal medium, sterile distilled water, and autoclaved sterile PDB were used as controls. Each treatment was replicated six times. The treated cell cultures were arranged in a completely random design and allowed to grow for another 10 days. At the end of the incubation period, the cell cultures were harvested and weighed (fresh weight). Data were analyzed using SAS.  Comparison of the effectiveness of cell free filtrate and emde homogenate of D. festucae to suppress the growth of fescue cell suspension cultures  In this experiment, only isolate no. 22170 (isolatedfromF. rubra host) was used. The cell-freefiltrateswere preparedfromaliquots that were used to prepare the crude homogenates. The fungal cultures were filtered using three layers of Whatman filter paper no. 41 without tissue homogenization, followed byfiltrationusing a 15 um fritted glass funnel on a suction flask. Four different treatments were compared. They included the cell-free filtrate, the crude homogenate, with sterile PDB and water as the control treatments. Each treatment was replicated ten times and arranged in a completely random design. Incubation conditions were as described for the previous experiment. After 12 days incubation, cell cultures were harvested and weighed (fresh weight). Data were analyzed using SAS.  43 Detemiination of the effect of concentration of fungal crude homogenates on the growth of fescue cell suspension cultures.  In this experiment, treatments were different concentrations of fungal crude homogenate of isolate nos. 22168, 22169 and 22170. Concentration of crude homogenate was defined as the volume of crude homogenate per 30 mlfinalvolume of the medium. The concentrations of fungal crude homogenate were 0, 6, 12, 18 and 24 ml per 30 ml final volume. Thefinalvolume and concentration of the medium constituents after addition of fungal crude homogenate were 30 ml and one half strength respectively. The pH of the media was adjusted to 5.8 before the addition of fungal crude homogenate. A possible change in the pH of the medium after treatment was considered as a potential contributing factor to every growth effect caused by the fungal filtrates. There were 15 combinations of media. Each combination was replicated five times. Fungalfiltrateand cell culture preparations, and cell harvesting were as described for the previous experiments. Cells were harvested at the time browning was observed in the cultures, and thefreshweight increase was measured.  Effects of fungal crude homogenate on the induction of PAL activity in cell cultures of creeping red fescue  Suspension cell cultures of'Boreal' were grown in 125 ml flasks containing 24 ml of one half strength MS basal medium. The cell cultures were allowed to grow for 3 days prior to challenge with a fungal crude homogenate of isolate no. 22170. Control cell cultures were challenged with sterile distilled water. The volume of  44 fungal crude homogenate or sterile distilled water added to each cell culture was 6 ml. Sampling of cell cultures for PAL assay occurred at 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 30 hours after inoculation. At each sampling, five cell cultures were taken as replicates. One gram of cells was takenfromeach culture for PAL assay. The cells werefrozenin liquid nitrogen immediately after sampling and stored at -20°C until assayed. The PAL assay was done using radiolabelled assay methods of Bernards and Lam (personal communication) based on that of Hanson and Havir (1981). The protocol of the PAL assay is presented in Appendix 4.  3. Results  Examination of the growth of D. festucae in PDB medium  Exponential phase of the fungal growth was achieved during days 6 to 9 after initial subculture (Figure 5). On day 10 , the fungal cultures reached stationary growth followed by mycelial death reflected in mycelial weight loss.  45  3.0  r  Figure 5. Mycelial growth of D. festucae initiatedfrom6 agar plugs (0.5 cm diameter) of mycelial colony in a 100 ml potato dextrose broth medium and incubated on a gyratory shaker at 100 rpm at room temperature of 25 ± 3°C and normal room lighting (5.7 umol s" m"). • =freshweight, = dry weight. 1  2  A  46 Determination of the effects of sterilization and sterilization method on the effect of fungal crude homogenates on the growth of fescue cell suspension cultures The impact of sterilization method on the comparison of treated (fungal filtrate) and untreated (control) cell suspension cultures is summarized in Figure 6 and Table 8. Crude homogenates of culture filtrates which were autoclaved showed significantly less cell growth suppression than the cold sterilized (0.22 urn Millipore filter) filtrates. The overall comparison of all treatments versus the "pooled" controls showed a significant treatment effect offiltrateon growth suppression. The differences observed within the control groups suggest the need for caution in interpreting these data. A comparison of filtrate treatments against the single PDB-treated control (the most appropriate) may show a lower magnitude effect. Isolate 22168 showed little evidence of suppression in this experiment; in fact, the autoclaved filtrate was stimulatory. This phenomenon may reflect the destruction of inhibitory compounds in the extract, and/or the release of nutrients or other growth stimulatory compounds. The suppression observed by isolate 22168 in prelirninary experiments and in the concentration experiment described in a later section suggested that this result may have been peculiar to this particular batch of extract. Comparison of the effectiveness of cell free filtrate and crude homogenate of D.  festucae to suppress the growth of fescue cell suspension cultures  No significant difference (P < 0.05) was observed between the effects of cellfreefiltrateand homogenized crude homogenate on cell growth suppression (Figure 7 and Table 9). Compared to the controls, however, both treatments significantly suppressed cell growth.  47  9  Media  Figure 6. Growth of fescue cell cultures in half strength M S basal media with added hot (H) or cold (C) sterilized culture filtrate of different isolates of D. festucae. The treatments of basal media + none, basal media + basal media, basal media + water, and basal media + PDB were used as controls. Each point represents the mean of 6 observations.  48 Table 8. Analysis of variance, orthogonal contrast and Tukey's Studentized Range (HSD) Test of data of cell fresh weight of fescue cell cultures grown in media containing different treatments of fungal filtrates of D. festucae isolate 22170. Mean Square  F Value  Pr>F  Source  df  Sum of Squares  Total  59  55.0202  Treatments  9  32.6286  3.6254  8.10**  0.0001  Controls (1-4) vs filtrates (5-10)  1  5.0074  5.0074  11.18**  0.0016  Within controls: 1 vs 2-4 2 vs 3,4 3 vs 4  1 1 1  3.5051 5.9382 3.1059  3.5051 5.9382 3.1059  - 7.83** 13.26** 6.94*  0.0073 0.0006 0.0112  Cold sterilization (5,7,9) vs hot sterilization (6,8,10) 1 7.2092  7.2092  16.10**  0.0002  Within cold sterilized filtrates: 5 vs 7,9 1 4.5128 7 vs 9 1 0.4041  4.5128 0.4041  10.08** 0.90  0.0026 0.3467  Within hot sterilized filtrates: 6 vs 8,10 1 2.5085 8 vs 10 1 0.4374  2.5085 0.4374  5.60* 0.98  0.0219 0.3278  Error  0.4478  50  22.3916  Note: number 1 to 10 in the 'Source' correspond to treatment listing as shown in the table below. *, ** =. significant at P < 0.05 and P < 0.01, respectively  Tukey's Studentized Range (HSD) Test for thefreshweight of cultured cells Alpha = 0.05, df=50, Mean Square Error = 0.447833 Critical Value of Studentized Range = 4.681 Minimum Significant Difference = 1.279 Means with the same letter are not significantly different. Tukey Grouping  B B B B B B B  A A A A D D D D D D  C C C C C C  Mean  N Treatments  8.7437 8.0965 8.0340 7.4955 7.3817 7.1137 7.0488 7.0165 6.5030 6.1360  6 6 6 6 6 6 6 6 6 6  N = number of replicates, H = hot sterilized filtrate, C = cold sterilized filtrate  2 = 6 = 3 = 10 = 5 = 8 = 1 = 4 = 9 = 7 =  sterile MS basal media isolate 22168 (H) sterile distilled water isolate 22170 (H) isolate 22168 (C) isolate 22169 (H) no additional treatment sterile PDB isolate 22170 (C) isolate 22169 (C)  50  12  C e l l - f r e e filtrate  C r u d e filtrate  PDB  Water  M e d i a treatments  Figure 7. Growth of fescue cell cultures in half strength MS basal media added with with cell-free or crude homogenates of D. festucae isolate 22170. The treatments of basal media + PDB and basal media + water were used as controls. Each point represents the mean of 10 observations.  51 Table 9. Analysis of variance, orthogonal contrast and Tukey's Studentized Range (HSD) Test of data of cellfreshweight of fescue cell cultures grown in media containing cell-free filtrates, crude homogenates of D. festucae isolate 22170, or controls Source  df  Type III SS  Mean Square  F Value  Pr > F  Total Replication Treatments  39 9 3  18.8514 1.4350 11.4350  0.1594 3.8117  0.72 0.6867 17.21** 0.0001  Filtrates (1, 2) vs controls (3, 4)  1  10.0000  10.0000  45.14** 0.0001  Between filtrates: 1 vs2  1  0.8570  0.8570  3.87  0.0596  Between controls: 3 vs 4  1  0.5780  0.5780  2.61  0.1179  Error  27  5.9814  0.2215  Note: numbers 1 to 4 in the 'Source' correspond to treatment listing as shown in the table below *, ** = significant at P < 0.05 and P < 0.01, respectively Tukey Grouping  Mean  N  Treatments  A A  10.073 9.733 9.110 8.696  10 10 10 10  4 = sterile distilled water 3 = sterile PDB 1 = cell-free filtrate 2 = crude homogenate  B B  N = number of replicates Means with the same letter are not significantly different.  52 Examination of cell growth inresponseto different concentrations of fungal crude homogenates  The level of cell growth suppression was dependent on the volume of fungal crude homogenate added to cell cultures; suppression was higher as concentrations of fungal filtrates were increased (Figure 8). The cells started to die when challenged with 6 ml or more of the fungal filtrate for all isolates tested. The cells were likely lysed, therefore resulting in cell weight loss. Browning of cells was first observed in those cultures challenged with 24 ml of filtrate on day 4 after treatment.  Observation of time course analysis of fungal crude homogenates on the induction of PAL activity in cell cultures  PAL activity was detectable in both treated and control cultures (Figure 9). In both cases, rates were extremely variable from one sampling time to the next. This resulted in a difficulty in data interpretation. In coping with this problem, a regression analysis was made to enable the detemiination and comparison of a likely trend of PAL activity in both treated and control cultures. The regression analysis showed no significant difference (P < 0.05) between the time course response of fungal filtratetreated versus control cultures (Appendix 3).  53  Figure 8. Growth suppression in fescue cell cultures in response to different concentrations of crude homogenate of 3 isolates of D. festucae. Each point represents the mean of 5 observations done at the end of an 8-dayincubation period. = isolate no. 22168, • = isolate no. 222169 and • = isolate no. 22170. A  54  0.03  r  0.00 I 0  1  1  1  1  1  1  1  1  1  1  3  6  9  12  15  18  21  24  27  30  Time of sampling (hours after inoculation)  Figure 9. Effect of D. festucae crude homogenate on the induction of PAL activity in fescue cell cultures. Each point represents the mean of 5 observations. • = dH 0 treated cell cultures, = fungal filtrate treated cell cultures. The straight lines represent the regression analysis for the control (dH 0) ( ) and the fungal filtrate treatments (—). Specific PAL activity was expressed in pKatal per ug extracted protein in each cell sample. The concentration of the total extracted protein in each sample was deteirnined, using Bradford method. A  2  2  55 4. Discussion  Growth of D. festucae in liquid PDB achieved its exponential phase during day 6 to 9 after initial subculture. It reached stationary maximum growth on day 10. If the growth suppression by D. festucae is caused by a fungal exudate, as reported for D. applanata  toxin (Suchuring and Salemink, 1972; and Broekoven et al., 1975), the  production of a putative toxin(s) is likely associated with the growing phase of the mycelium. In cultures of Phytophthora cactorum, toxin production was also associated with the mycelial growth (Plich and Rudnicki, 1979). Using this assumption, maximum concentration of a putative toxin in liquid culture of D. festucae was likely achieved by day 10. Therefore, 8 to 9 day old fungal cultures were used for routine production of crude homogenates and cell-free filtrates throughout the experiments. Challenging cell cultures of creeping red fescue with crude homogenates of D. festucae  resulted in cell growth suppression. Suppression was observed not only in  response to isolate no. 22170 isolatedfromF. rubra but also with isolate no. 22169 isolatedfromF. scabrella These results are consistent with those of previous tests of pathogenicity. In a cross-infection test, Smith and Shoemaker (1974) observed that eleven isolates of P. idahoensis derivedfromsix different species of Festucae were pathogenic on all hosts tested. The only difference reported in these tests was in the level of virulence. The effect of crude homogenates of isolate no. 22168 on the growth of fescue cell suspension cultures was not consistent. In the second experiment, crude  56 homogenates of this isolate did not suppress the growth of fescue cell suspension cultures; while in the other experiments, it suppressed cell culture growth. The inconsistent effect may be a consequence of the variable quantity of mycelium on agar plugs used for fungal culture preparation in every experiment. Because of this reason, fungal filtrates of the same volume might have contained different concentrations of the putative fungal toxin. Inoculum quantification was recognized as the main problem of the present studies since attempts to induce sporulation in D. festucae were not successful. Compared to the growth of fungal medium (PDB)-treated cell cultures, the growth of cell cultures challenged with fungal crude homogenates was significantly reduced. The growth of cell cultures challenged with sterile PDB was similar to the growth of cell cultures which were not challenged. Such observations provide circumstantial evidence that the observed cell growth suppression was a response to the presence of a toxic compound(s) in the fungal crude homogenate. This compound might be fungal exudate that was produced in planta as well as in vitro, a cell wall component (elicitor), or a fungal metabolite that was produced only in axenic culture. The first two possibilities were examined in a separate experiment and are discussed in the next paragraph. The third possibility receives some supportfromthe observation that some fungi produce metabolites in artificial media, but not in planta. Vardi et al. (1986) found that the use of Phytophthora citrophthora culture filtrate as a reliable selection tool for citrus calli was doubtful since this fungus produced 2,4-D in culture. The presence of 2,4-D was suspected to be the reason why the growth of calli of  57 susceptible citrus was not significantly reduced when challenged with P.  citrophthora  culture filtrate. Similarly, Yoder (1983) stated that culture filtrates cannot necessarily be expected to select plants resistant to disease since growth media colonized by microorganisms contain many secondary metabolites which, in combination, can be phytotoxic but have nothing to do with disease development. Comparison of the effects of cellfreefiltrateand crude homogenate showed no significant difference on cell growth. It has been reported that some phytotoxic compounds, such as those of Fusarium oxysporum f. sp. dianthi race 2 (Buiatti et  al.,  1985) and of Phytophthora infestans (Doke and Tomiyama, 1980) are cell wall components (elicitors); while the others such as those (Vardi et  al.,  1986) and of Didymella  applanata  of[Phytophthora citrophthora  (Suchuring and Salemink, 1972; van  Broekoven et al., 1975) are fungal exudates. Cell-wall-components (elicitors) usually can only be extracted by tissue homogenization. In this experiment, homogenized fungal cultures (crude homogenates) did not result in any increase in cell growth suppression. Therefore, the putative phytotoxic compound(s) was unlikely a cell wall component (elicitor). Thus, besides the possibility that a putative toxic compound produced by D. festucae is a fungal metabolite that is produced only when the fungus is grown in axenic cultures, it might be a fungal exudate specific to the disease process as in the case of D.  applanata  toxin.  D. applanata  had been reported to injure  raspberry plants by releasing a glycopeptide phytotoxin in the host plant tissue (van Broekoven et  al.,  1975).  D. festucae also  in PDB (Smith et al., 1968).  produced a pigmented exudate on PDA and  58 Autoclaving eliminated the ability of fungal crude homogenates to cause cell growth suppression. If growth suppression was a consequence of the presence of a D. festucae  toxic compound, this toxic compound must be a heat sensitive compound. It  is difficult to speculate further on the chemical properties of this phytotoxic compound based solely on its sensitivity to heat treatment; structures of phytotoxins include an array of chemical substances such as glycosidic-bond, terpenoid-origin, or amino acidderived compounds (Strobel, 1974). Nevertheless, this result provides information on how to prepare culturefiltrateof D. festucae when its phytotoxic effect is to be retained. Increasing concentrations of fungal crude homogenates resulted in cell growth suppression and eventual cell death reflected in cell weight loss due likely to a cell lysis. The levels of suppression observed, however, should not be taken as a general quantitative response of fescue cell suspension cultures to a specific concentration of D. festucae culture filtrate.  Under these growth conditions, the concentration of the  putative toxic compound contained in a volume of fungal filtrate was not necessarily reproducible. The results of PAL assay showed that regardless of the magnitude of PAL activity, no significant difference (P < 0.05) was observed between PAL activity in fungalfiltrate-treatedand water-treated (control) cell cultures. Thefirstexplanation of the insignificant difference between the PAL activity in culturefiltrate-and watertreated cell cultures may be because the cell cultures were derivedfroma susceptible cultivar. This conclusion is based on existing evidence showing an insignificant  59 difference in PAL activity between susceptible plant tissues challenged with fungal toxins and those challenged with control treatments (water) (Bhattacharyya and Ward, 1988; Cahill and McComb, 1992; and Chorchete et al., 1993). In a susceptible tissue, an irregular variation in time course PAL activity is also not uncommon, and is usually ignored (Bhattacharyya and Ward, 1988; and Cahill and McComb, 1992). This argument, however, does not justify the use of PAL activity to determine the response of creeping red fescue cell cultures to D. festucae culture filtrate, unless a comparison study using resistant and susceptible cultivars of creeping red fescue confirms the above argument. The second explanation is that PAL might not be the critical enzyme function confening the defense reaction to D. festucae in creeping red fescue. In grasses, pcoumaric acid, a precursor of a variety of natural products that function in defense mechanisms, may be produced via two different pathways. The first pathway is intermediated by the formation of L-phenylalanine, while the second one is intermediated by the formation of L-tyrosine. Both L-phenylalanine and L-tyrosine are then converted by phenylalanine/tyrosine ammonia-lyase into /»-coumariC acid (Neisch, 1961; Jangaard, 1973; Geoffrey, 1988). While both pathways exist, PAL activity appears to be the predominant route (Jangaard, 1973).  GENERAL DISCUSSION  Initial studies on the establishment and maintenance of callus and cell cultures of creeping red fescue resulted in the differentiation of three types of callus tissue. Among these three types, only type 1 and type 3 calli could be used as sources for callus/cell culture materials and/or plantlet regeneration. With the exception of cultivar 'Barcrown', type 1 calli of 'Boreal' and 'Cindy' were non-regenerable, while the type 3 calli were regenerative. Most calli of 'Barcrown' were regenerative since shoot-producing calli were observed among type 1 calli of this cultivar. These results suggest that cultivar and genotype sources are important determinants of the callus type obtained in the establishment of callus and/or cell cultures of creeping red fescue. Not all cell suspension cultures of creeping red fescue can be used effectively in the study of the  in vitro  behavior of creeping red fescue-D. festucae interaction.  Only those derivedfromtype 1 callus of 'Boreal' and 'Cindy' were effective for the above purpose. The more uniform cell population of these cell cultures facilitated exposure to fungal filtrates added to the medium. Therefore, for the purpose of in vitro  study of the creeping red fescue-D. festucae interaction, cell cultures derived  from type 1 calli of 'Boreal' and 'Cindy' are more appropriate. The study of the creeping red fescue-D. festucae interaction may be done using a cell culture-fungal filtrate system. Culturefiltratesof D. festucae were found to be capable of suppressing the growth of fescue cell suspension cultures, but not with the control treatments. The effects of culture filtrates of the three isolates tested were  60  61  similar and consistent throughout the present experiments. This may indicate that culture filtrates of these isolates have the same biochemical properties in vitro, which may not reflect in vivo host specificity differences. Cell growth suppression was very likely due to the presence of a toxic compound in the culture filtrate. This compound was unlikely to be a cell wall component. However, it has not been detennined whether the toxic compound is a fungal exudate, or is a fungal metabolite produced only in axenic culture. Further detailed study is required to characterize this compound. In addition, it is necessary to detenriine whether the toxic compound is involved in disease development. This is because not all phytotoxins are important in disease development. The use of agar plugs of mycelium as inocula resulted in a non-reproducible concentration of putative toxin as reflected in an inconsistent quantitative effect of fungal filtrate on cell culture growth. The use of spores as inoculum was precluded due to the difficulty in inducing sporulation. Therefore, the use of purified putative phytotoxin might be the most effective solution to the above problem. The use of PAL assay as a biochemical marker of the defense mechanism in fescue cell cultures against D. festucae culturefiltratesseemed to be ineffective. There was no difference in time course PAL activity between fungal filtrate- and water-treated cell cultures. It was argued that this might be because the cell cultures were derivedfroma susceptible cultivar. The problem became more confounded by the fact that in grass species, />coumaric acid, the precursor of natural products, not only convertedfromL-phenylalanine by PAL but may also have been converted from  62  L-tyrosine by TAL. The problem with attempts to determine the biochemical response of fescue cell cultures against D. festucae culture filtrates is that no field grown creeping red fescue cultivar is known to be resistant to stem eyespot disease. The cultivar 'Boreal' used to initiate cell cultures in the present study is generally susceptible to stem eyespot under field conditions. Consequently, even if PAL activity was more enhanced in fungalfiltrate-treatedcell cultures than that in controls, it did not necessarily mean that PAL activity can be potentially used as a unique marker of defense mechanism of the fescue cell culture against D. festucae fungal filtrate. Determination of whether PAL activity is a potential marker will require confirmation through multiple cultivar tests.  CONCLUSIONS  Based on the results of the experiments carried out in the present studies, the following conclusions were drawn. 1. There were three types of calli initiatedfromcaryopses of creeping red fescue cultivars 'Boreal', 'Cindy' and 'Barcrown'. The type of callus was dependent on the cultivar and genotype sources used for callus initiation. Type 1 was nonregenerable, type 3 was regenerative, while type 2 was not grouped as nonregenerable nor as regenerative due to its retarded growth. 2. Among the initiated calli, only type 1 of 'Boreal' and 'Cindy' that could be used effectively for the preparation of homogenous cell suspension cultures for in  vitro  study of creeping red fescue-D. festucae interaction. 3. Culture filtrates of D. festucae isolate nos. 22168 and 22169 (both isolatedfromF. scabrella),  and 22170 (isolatedfromF. rubra) could be used to challenge fescue  cell suspension cultures and resulted in cell growth suppression. Among culture filtrates of the above isolates, no difference was observed in their ability to cause cell growth suppression in fescue cell suspension cultures. 4. 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Endogenous auxins in healthy and disease plants. In Encyclopedia of plant physiology. Edited by: R. Heitfuss, P. H. Williams. New series, vol. 4. Springer. Berlin, pp: 560-581. Plich, M and Rudnicki, R. M 1979. Studies of the toxins of Phytophthora cactorum pathogenic to apple trees. Phytopathol. Z. 94: 270-278. Smith, J. D. 1971. Phleospora idahoensis on native Festuca spp. in the northwestern Great Plains. Can. J. Bot. 49: 377-381. Smith, J. D, Elliott, C. R. and Shoemaker, R. A. 1968. A stem eyespot of red fescue in northern Alberta. Can. Plant Dis. Surv. 48: 115-119. Smith, J. D. and Elliott, C. R. 1970. Stem eyespot on introduced Festuca spp. in Alberta and British Columbia. Can. Plant Dis. Surv. 50: 84-87.  68 Smith, J. D. and Shoemaker, R. A. 1974. Didymella festucae and its imperfect state, Phleospora idahoensis, on Festuca species in western North America. Can. J. Bot. 52: 2061-2079. Strobel, G. A. 1974. Phytotoxins produced by plant parasites. Annual Review of Plant Physiology. Edited by: W. R. Briggs, P. B. Green and R. L. Jones. 25: 541-566. Suchuring, F. and Salemink, C. A. 1972. A phytotoxinfromDidymella applanata cultures. In Microbial Toxins. Edited by: Kadis et al., Vol. VIII. Academic Press. New York, pp: 193-209. Torello, W.A and Symington, A. G. 1984. Regenerationfromperennial ryegrass (Lolium perenne L.) callus tissue. HortScience 1: 56-57. Torello, W. A , Symington, A. Gand Rufrier, R. 1984. Callus initiation, plant regeneration, and evidence of somatic embryogenesis in red fescue. Crop Science 24: 1037-1040. Torello, W. A., Rufrier, R. and Symington, A. G. 1985. The ontogeny of somatic embryosfromlong-term callus cultures of red fescue. HortScience 20: 938-942. van Broekhoven, L.W., Minderhoud, L., Holland, G. J. J., Tausk, R. J. M. and Lousberg, R. J. J. Ch. 1975. Purification and properties of a phytotoxic glycopeptidefromDidymella applanata (Niessl) Sacc. Phytopathol. Z. 83: 49-56. Vardi, A , Epstein, E. and Breiman, A. 1986. Is the Phytophthora citrophthora culture filtrate a reliable tool for the in vitro selection of resistant citrus variants?. Theor. Appl. Genet. 72: 569-574 Wang, D-Y, and Vasil, I. K. 1982. Somatic embryogenesis and plant regeneration from inflorescence segments of Pennisetum purpureum Schum. (Napier or Elephant grass). Plant Sci. Lett. 25: 147-154. Yoder, O. C. 1983. Use of pathogen-produced toxin in genetic engineering of plants and pathogens. In Genetic engineering of plants. Edited by: T. Kosuge, C P . Meredith, A. Holaende. Basic Life Science. Vol. 26. Plenum Press. New York, pp: 335-353. Zaghmout, O. M F. and Torello, W. A. 1988. Enhanced regeneration in long-term callus cultures of red fescue by pretreatment with activated charcoal. HortScience 23: 615-616.  69 , 1989. Somatic embryogenesis and plant regenerationfromsuspension cultures of red fescue. Crop Sci. 29: 815-817. . 1990. Isolation and culture of protoplast from embryogenic suspension cultures of red fescue (Festuca rubra L.). Plant Cell Reports 9: 340-343. _ . 1992. Restoration of regeneration potential of long-term cultures of red fescue (Festuca rubra L.) by elevated sucrose levels. Plant Cell Reports 11: 142-145.  70  APPENDICES  71  1. Ingredients of MS Basal medium  NH4N0 KN0 CaCl .2H 0 MgS0 .7H 0 KH P0  1650 1900 440 370 170  3  3  2  2  4  2  2  4  37.3 27.8  H3BO3  6.2 mg/1 22.3 mg/1 8.6 mg/1 0.83 mg/1 0.25 mg/1 0.025 mg/1 0.025 mg/1  4  2  MnS0 .4H 0 ZnS0 .7H 0 KI Na Mo0 .2H 0 CuS0 .5H 0 CoCl .6H 0 4  2  4  2  2  4  2  4  2  2  2  Thiarnine HC1 Myo-inositol Nicotinic Acid Pyridoxin HC1 Kinetin 2,4-D IBA *) Sucrose **) Agar ***)  \  mg/1 mg/1  Na EDTA FeS0 .7H 0 2  *) **) ***)  mg/1 mg/1 mg/1 mg/1 mg/1  0.1 100 0.5 0.5 0.1 5  mg/1 mg/1 mg/1 mg/1 mg/1 mg/1  0.1  mg/1  30000 12000  mg/1 mg/1  : used only for rooting media : sucrose of 30 g/1 was used for any media regardless of their strength : only used to make solid media  72 2. Composition and strength of media for culture maintenance  MS BASAL  VITAMINS  HORMONE  1. Callus culture  Full Strength + 12 g/1 agar + 30 g/1 sucrose  Thiamine HC1 Nicotinic acid Pyridoxin HC1  2,4-D Kinetin  2. Cell culture  Half Strength + 15 g/1 sucrose agar free  Thiamine HC1 Nicotinic acid Pyridoxin HC1  2,4-D Kinetin  3. Plant regeneration (shoot initiation)  Half Strength + 15 g/1 sucrose + 12 g/1 agar  Thiamine HC1 Nicotinic acid Pyridoxin HC1  4. Root initiation  Half Strength + 15 g/1 sucrose + 12 g/1 agar  TWamine HC1 Nicotinic acid Pyridoxin HC1  /  IBA  73 3. Regression analysis of time course P A L activity  E:  X  X  0 3 6 9 12 15 18 21 24 27 30  0 9 36 81 144 225 324 441 576 729 900  165  3465  Average: 15  2  Y,  Y,  0.021 0.024 0.009 0.007 0.011 0.022 0.008 0.012 0.024 0.012 0.015  0.165  0.015  XY,  Y  0.000441 0.000576 0.000081 0.000049 0.000121 0.000484 0.000064 0.000144 0.000576 0.000144 0.000225  0 0.072 0.054 0.063 0.132 0.33 0.144 0.252 0.576 0.324 0.45  0.014 0.016 0.023 0.021 0.019 0.017 0.018 0.022 0.025 0.011 0.016  0.000196 0.000256 0.000529 0.000441 0.000361 0.000289 0.000324 0.000484 0.000625 0.000121 0.000256  0 0.048 0.138 0.189 0.228 0.255 0.324 0.462 0.6 0.297 0.48  0.002905  2.397  0.202  0.003882  3.021  2  2  0.018  Y  2 2  XY  2  Regression analysis of PAL activity: PAL activity of dH20 treated: Multiple R R Square Adjusted R Square Standard Error Observations  0.119548127 0.014291755 -0.095231384 0.006862576 11  Analysis of Variance: df Regression 1 Residual 9 Total 10  Sum of Squares 6.14545E-06 0.000423855 0.00043  Mean Square 6.14545E-06 4.70949E-05  F Significance 0.130490734 0.726258275  Coefficients Standard Error t Statistic P-value Intercept 0.016181818 0.003871015 4.180252062 0.001886954 xl -7.87879E-05 0.000218107 -0.361235013 0.725438927  Lower 95% Upper 95% 0.007424967 0.024938669 -0.00057218 0.000414605  PAL activity of fungal filtrate treated: Multiple R R Square Adjusted R Square Standard Error Observations  0.021775751 0.000474183 -0.110584241 0.004377514 11  Analysis of Variance: df Regression 1 Residual 9 Total 10  Sum of Squares 8.18182E-08 0.000172464 0.000172545  Mean Square 8.18182E-08 1.91626E-05  0.004269675  Significance F 0.949329527  Coefficients Standard Error t Statistic P-value Lower 95% Intercept 0.0185 0.002469251 7.492151213 2.08148E-05 0.01214162 xl -9.09091E-06 0.000139127 -0.065342748 0.949188949 -0.00032382  Upper 95% 0.024085838 0.000305635  Regression equation of PAL activity dH 0 treated: 7, = 0.0162 - 0.0000787879 X Regression equation of PAL activity fungal filtrate treated: Y = 0.018 - 0.0000909091 X 2  2  75  Test for slope difference of two regression lines:  J.x  =T.X -  2  2  -  { Y  n  Z y, = Z ^ - ^ 2  2  Z*y, = Z AT, -  = 3465--^-=990 11  X )  '  F  )  = 0.002905 -  2  n  *  1  ZK |  «  = 2.397-  1  0  1  6  5  2  1  6  11 6  5  x0  = 0.00043  5  11  = -Q.Q78  6, = IL^J. = ZMTI _ .00007879 Z* 990 =  2  Iy  2 2  = I ^2 '2 ~ ^ 2  7  0  ^ = 0.003882 -  0  2  0  y  = ZX7 -  2  *  Z  2  Z  2  y  n  -0,009  Z*  = 0.0001725  2  11  n Tx  2  =  » . 3.021-  1  6  5  x0  2  0  Q  j < * = 0-000423855 + 0.000172464 residual DF + residual DF 9+ 9  resi  ual  SS  +  r e s i d u a l  SS  I  j2MS  f2x 0.000033128  _  Sb,-b, v v v2 1 "2 V i *  V"V  2  b  = -0.009  990  Residual MS =  = i~ 2 Sb-b  2  11  990  2  0.0002587  (-0 00007879)-(-0.00000909) 0.0002587  b  =  0.05,(2),v=18 =  ?  t  2  ~  1  0  =  0  ,,  Q  4  1  0.05,(2),v=18  ?  => the two regression lines have the same slopes  =  Q  0  0  0  Q  3  3  m  76  Test for elevation difference of two regression lines:  SS of X for 'common regression' = 4. =2(Sx ) = 1980 2  Sum of crossproducts for 'common regression' = B =(£xy) +(Lxy)2 c  SS of Y for 'common regression' = C =(Zy \M£y ) 2  = 0.00043+0.0001725 = 0.0006025  2  c  = -0.078 +(-0.09) = -0.087  l  2  D 2  Residual SS for 'common regression'= SS . = Q . — ^ = 0.0005987 C  Residual DF for 'common regression'=DF = 2 « - 3 = 19 C  Residual MS for 'common regression' = (S  )  2 YJ{  C  =  =  DF B _ -0-087 c  Ac~ 1980  e  ^0.05,(2),v = 1 9 - -  t  <  19  = 0,00003151  = 0.00004394  (Yi-Y2)-b (Xi-X2)  2  C  a 0 0 Q 5 9 8 7  = -0.39635  0 9 3  t,•0.05,(2),v = 19  => the two regression lines have the same elevations  Deterrrwiation of common regression lines:  common regression coefficient = b = 0.00004394 c  common Y intercept = a = Y c  _ n^Xx+n-tXi  P  bX c  p  _ ^  n\ + nj y _ nji + n Y _ (11 x 0.015)+(ll x 0.018) _ 2  « i  +  «2  2  Q  1  6  5  22  a = 0.0165 -(0.00004394 x 15)= 0.01584 c  A  The common regression equation: Y = 0.01584-0.00004394^  78 4. Radiolabel Assay for Phenylalanine Ammonia-lyase (E.C 4.3.1.5) at the Microfuge level  1. PAL stock reagents/solutions:  - 100raML-Phenylalanine stock (unlabelled) stored at 4°C - 20 uCi L-[U- C]-Phenylalanine, concentration = 50 LrCi/ml, 14  specific activity = 495 mCi/mmol (ICN) - glass distilled water 2. PAL substrate preparation:  PAL substrate solution contained 1.0 ml 100 mM L-phe (unlabelled), 0.2 ml L-[U- C]-Phe, and 8.8 ml glass distilled water. Thefinalconcentration of L-Phe 14  in PAL substrate was 0.5 umol/50 ul substrate (or 10 mN L-Phe). The PAL substrate was stored in an isotopefreezercompartment. The total activity of the PAL substrate was 111.252 dpm/assay (50 ul substrate/assay). 3. PAL assay solutions:  - Extraction buffer: 200 mM Tris-acetate, pH 7.5; 5 mM 2-Mercaptoethanol was added on day of use since 2-Mercaptoethanol oxidizes on standing, particularly in Tris buffers. The extraction buffer stock solution was stored at 4°C. - Assay buffer: 100 mM Potassium Borate, pH 8.7; 5 mM 2-Mercaptoethanol was also added on day of use. The assay buffer stock solution was stored at 4°C. - 4 M H S0 2  4  - 266 mM cinnamic acid carrier - Toluene : ethyl acetate (1:1)  79 4. Penefsky columns packing:  - Biogel P6DG resin (Biorad) swollen in assay buffer (100 mM Potassium Borate, pH 8.7) was used as the beads - Penefsky column was madefrom3 ml syringe without needle or plunger. Three layers of miracloth were used to cover the bottom end of the syringe - the resin was loaded and compacted into the syringe to make a final bead volume of 2.5 ml. 5. Extraction and desalting of soluble proteins (done at 4°Q:  - 1 g cell samples were ground in the presence of 1 ml extraction buffer soaked with PVP and liquid nitrogen - samples were transferred to a 2 ml centrifuge tube to be thawed and vortexed - after centrifugation at 10,000 g for 5 minutes at 4°C, 800 ml of the supernatant was transferred to the column for desalting - the protein extract was allowed to saturate the bead, and then spun in a clinical centrifuge at 15,000 g for 1 minute at 4°C to get the desalted protein - the final volume of the desalted protein was adjusted to 1 ml with assay buffer; and kept on ice until reaction. 6. PAL assay for the desalted protein:  - four samples each of 150 ul aliquot were prepared for PAL assay in a 1.5 ml centrifuge tube, and the remainder was reserved for Bradford assay - one sample was boiled in a water bath for 30 minutes to generate the control - to each tube, 50 ul PAL substrate was added using a repeator pipettor and  80 vortexed briefly to mix. - the tubes were incubated in 30°C in a waterbath for two hours. - the reaction was stopped by adding 50 ul 4 M H S0 and 10 ul cinnamic acid 2  4  carrier to each tube using a repeator pipettor - 1 ml toluene: ethyl acetate was used to extract the organic phases. - 5 ml scintillation cocktail was added to 500 ul organic phase in a clean capped scintillation vial - the vials were loaded into a rack for radioactivity reading using a Beckman scintillation counter; in addition, five replicates of each 5 ul aliquot of PAL substrate alone was also included to get the dpm/mol L-Phe. 7. Determination of total protein concentration using Bradford method:  - to make Bradford reagent; 50 ml 95% ethanol, 100 mg coomassie brilliant blue G-250 (Kodak), 100 ml (85%) phosphoric acid, and distilled water (to make a 1000 ml final stock solution) were mixed together, filtered by gravity through fluted Whatman # 4 filter paper, and stored in a dark dispensing glass bottle - the regression line of absorbances versus concentrations of Bovine serum albumin (BSA) was used as standard curve - the concentrations of BSA used to make the standard curve were: 1, 2, 5, 10, 15, and 20 ug/ul; the absorbances were measured at X of 595 nm - 20 ul soluble desalted protein and 50 ul 1 M NaOH were pipetted into a 1.5 ml centrifuge tube, and added with 1 ml Bradford reagent; the blank contained 20 ul distilled water, 50 ul 1 M NaOH, and 1 ml Bradford reagent  81 - the absorbances of the prepared samples were measured at 595 nm, and the protein concentration was determined based on the standard curve. P A L activity calculation:  - total dpm (disintegration per minute) is calculatedfromthe average value of five replicates of 5 ul aliquot of substrate. Since 50 ul of PAL substrate was used, and this represented 0.5 umol of L-Phe, the total dpm  = (the average dpm of 5 ul aliquot x 10) / 0.5 umol L-Phe  if the average dpm of 5 ul aliquot = A dpm, total dpm  = 20A dpm/umol L-Phe = 20A x 10 dpm/mol L-Phe 6  - total P A L activity is expressed as pKatal, where one Katal defines the conversion of one mole of substrate in one second. net dpm counted  = the difference between the dpm for each sample (i.e. the average of triplicates) and that for boiled controls (i.e. the average of all boiled controls)  if the net dpm counted = B dpm reaction time  = 2 hours  net dpm  = (B dpm)/2 hour = (B/2) dpm/hour = (B/7200) dpm/second  total P A L activity = [(B/7200) dpm/second] / (20A x 10 dpm/mol L-Phe) 6  = (B/7200) / (20A x 10) mol L-Phe/second 6  82 = [(B/7200) / (20A x 10)] x 10 pmol L-Phe/second 6  12  = (B x 10) / (144 x A ) pKatal 3  specific PAL activity = total PAL activity / total protein concentration in 150 ul aliquot = PAL activity per jig protein  

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