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Carbon dioxide enrichment and the role of carbohydrate reserves in root growth potential of cold-stored… Chomba, Bernard Malata 1992

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CARBON DIOXIDE ENRICHMENT AND THE ROLE OF CARBOHYDRATERESERVES IN ROOT GROWTH POTENTIAL OF COLD-STORED ENGELMANNSPRUCE (PICEA ENGELMANNII PARRY) SEEDLINGSbyBernard Malata ChombaB.Sc.(For.) (Hons.) University of Dar-es-Salaam, TanzaniaA THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(DEPARTMENT OF FOREST SCIENCES)We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAApril 1992© Bernard Malata Chomba, 1992In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission. Department of^ ct^cF- sThe University of British ColumbiaVancouver, CanadaDate  AT^CY)-DE-6 (2/88)iiABSTRACTTwo experiments were conducted to examine the role ofcarbohydrate reserves in spring root growth potential (RGP)of Engelmann spruce (Picea engelmannii Parry) seedlings. Inthe first experiment, the effects of pre-storage carbondioxide enrichment (CE) on total non-structuralcarbohydrates (TNC) and post-storage root growth werestudied. Seedlings were grown from seed for 202 days ingrowth chambers with ambient (340 gL•L -1 ) and CO2 enriched(1000 gL•L -1 ) air. Reciprocal transfers between treatmentstook place at 60 and 120 days. Photoperiod was reduced at100 days to induce bud set. After 180 days seedlings werehardened-off for storage at -5 °C. At 268 and 327 days,seedlings were planted in a growth chamber in three waterbaths. New roots >5 mm long were counted after 28 daysgrowth. Seedlings were also assessed for bud break every twodays. At each planting time, and at 80, 120, 140, and 202days, seedlings were randomly selected from each of the CO2treatments and harvested for analysis of starch and solublesugar content. Growth data were also collected.In the second experiment, the relative contributions ofreserve carbon and current photosynthate to new root growthwere studied. Seedlings were raised using standard nurseryprocedures up to bud set (end of September, 1990). Seedlingswere then moved into a growth chamber and placed in fourPlexiglas boxes for stable carbon isotope labelling. Twoboxes received ambient CO2 with normal isotopic compositioniiiand the other two received air that was first stripped ofCO2 before adding back tank CO2 depleted in 13C. On November30, seedlings were hardened-off for storage for four monthsat -5 °C. Samples of seedlings were taken both before andafter storage and analyzed for carbohydrate reserves. Afterstorage, seedlings were planted in a growth chamber for 36days, during which time seedlings were sampled for new rootgrowth at 9, 18, and 36 days. Extracted carbohydrates andnew white roots were then analyzed for 13C/ 12C ratios.Carbon dioxide enrichment increased seedling biomassand root collar diameter, more so after bud set. Stem heightwas only slightly affected by CE. Shoot:root ratios (DWbasis) were not affected by CE, but decreased steadily withage. Carbon dioxide enrichment had little effect on solublesugars, starch, and TNC prior to bud set. After bud set, CEgreatly promoted the rate of reserve accumulation, but didnot influence the final level attained prior to storage.Needles were the major storage organ for soluble sugars,while roots were so for starch. Soluble sugars were notstrongly affected by the two and four months storage. Incontrast, there was more than 50% reduction in starch inneedles, stems, and roots, and almost a one-third loss ofTNC during storage. None of the CO2 treatments had anyinfluence on bud break and RGP. The isotope labellingexperiment indicated that carbohydrate reserves did make animportant contribution to spring root growth, but this sinkwas only a minor drain on the reserve pool.TABLE OF CONTENTSPageABSTRACT^Table of Contents^ ivList of Tables viList of Figures^ viiList of Appendices ixAbbreviations^Acknowledgements xiDedication^ xii1. INTRODUCTION^ 12. LITERATURE REVIEW 42.1. Carbohydrate Reserves and Root Growth^ 42.2. Carbohydrate Reserves and CO2 Enrichment^ 82.3. Carbohydrate Reserves and Root GrowthPotential (RGP) During Storage^ 102.4. Stable Carbon Isotopes as Tracers 122.5. Soil Temperature and Root Growth 132.6. Objectives^ 153. MATERIALS AND METHODS 163.1. Experiment One: Carbon Dioxide Enrichment(CE) and Carbohydrates^ 163.1.1. Seedling Production and Cold-storage ^ 163.1.2. Post Cold-storage Planting^ 193.1.3. Carbohydrate Analyses 223.1.3.1. Soluble Sugars Analysis byAnthrone Reagent^ 223.1.3.2. Starch Analysis by EnzymaticHydrolysis^ 233.2. Experiment Two: Stable Carbon Isotopesas Tracers^ 243.2.1. Seedling Production and Cold-storage ^ 243.2.2. Post Cold-storage Planting^ 273.2.3. Stable Carbon Isotope Analyses^ 293.2.3.1. New White Roots 313.2.3.2. Starch Extraction^ 323.2.3.3. Soluble Sugars Extraction^ 343.3. Experimental Design and Data Analysis 353.3.1. Experiment One^ 35iv3.3.2. Experiment Two^ 374. RESULTS^ 384.1. Carbon Dioxide Enrichment (CE) Experiment ^ 384.1.1. Effects of CE on Growth Prior toStorage^ 384.1.1.1. Biomass Accretion andPartitioning^ 384.1.1.2. Root Collar Diameter andHeight Growth 444.1.2. Carbohydrate Reserves 464.1.3. Bud break and Root Growth Potential ^ 574.2. Stable Carbon Isotopes as Tracers^ 605. DISCUSSION^ 655.1. Biomass Accretion and Partitioning^ 655.2. Root Collar Diameter and Stem Height 675.3. Carbohydrate Reserves^ 685.4. Effects of Cold-storage on CarbohydrateReserves^ 725.5. Bud Break 775.6. Root Growth Potential (RGP) ^ 805.6.1. Effects of CE and Soil Temperatureon RGP^ 805.6.2. Effects of Cold-storage on RGP^ 825.6.3. Role of Carbohydrates in RGP 846. CONCLUSIONS^ 887. RECOMMENDATIONS 908. LITERATURE CITED^ 93APPENDICES^ 103LIST OF TABLESTable^ Page1. The effects of CO2 enrichment (CE) on days to firstbud break in Engelmann spruce seedlings^ 582. The effects of CE on post-storage root growthpotential (number of new roots > 5 mm long) inEngelmann spruce seedlings^ 593. Isotopic composition (expressed as 8 13C value) ofwhole seedling total tissue and TNC before andafter storage^ 614. Trends in isotopic composition (expressed as813C value) of new white roots, and calculated percent of reserve carbon to new root construction of"labelled" seedlings 62viLIST OF FIGURESFigure^ Page1. A general view of the CE experiment during theseedling production phase^ 172. A general view of the pot arrangement for the CEexperiment in the growth chamber in one of thewater baths^ 213. A schematic diagram of the equipment used in thestable carbon isotope labelling experiment^ 254. General views of the arrangement when seedlings weremoved into the Plexiglas boxes for isotope labelling(a), and during post-storage planting (b) ^ 285. Effects of CE on (a) needle dry weight, and (b)stem dry weight of Engelmann spruce seedlings^ 406. Effects of CE on (a) root dry weight, and (b)total seedling biomass of Engelmann spruceseedlings^ 427. Effects of CE on shoot:root ratio (DW basis) ofEngelmann spruce seedlings^ 438. Effects of CE on seedling mean diameter (a), andstem height (b) of Engelmann spruce seedlings^ 459. Trends in whole plant TNC content of Engelmannspruce seedlings^ 4710. Trends in allocation of reserves to individualplant parts expressed as a percentage of themaximum TNC observed prior to storage^ 4811. Trends in soluble sugar content of needles (plusbuds) of Engelmann spruce seedlings 5012. Trends in the soluble sugar content of stems ofEngelmann spruce seedlings^ 5113. Trends in soluble sugar content of roots ofEngelmann spruce seedlings 5214. Trends in starch content of needles (plus buds)of Engelmann spruce seedlings^ 5415. Trends in starch content of stems of Engelmannspruce seedlings^ 55viiviii16. Trends in starch content of roots of Engelmannspruce seedlings^ 5617. Glucose (a), and starch (b) standard curves usedin carbohydrate analyses^ 120LIST OF APPENDICESAppendix^ Page1. ANOVA for the effect of CE on biomass partitioningin Engelmann spruce seedlings^ 1032. ANOVA for the effect of CE on total seedlingbiomass production in Engelmann spruce seedlings^ 1053. ANOVA for the effect of CE on shoot:root ratio inEngelmann spruce seedlings^ 1064. ANOVA for the effect of CE on diameter and stemheight in Engelmann spruce seedlings^ 1075. ANOVA for the effect of CE on carbohydrate reservesin Engelmann spruce seedlings at 80 days^ 1086. ANOVA for the effect of CE on carbohydrate reservesin Engelmann spruce seedlings at 120 days^ 1097. ANOVA for the effect of CE on carbohydrate reservesin Engelmann spruce seedlings at 140 days^ 1108. ANOVA for the effect of CE on carbohydrate reservesin Engelmann spruce seedlings at 202 days^ 1119. ANOVA for the effect of CE on carbohydrate reservesin Engelmann spruce seedlings at 268 days^ 11210. ANOVA for the effect of CE on carbohydrate reservesin Engelmann spruce seedlings at 327 days^ 11411. ANOVA for the effect of CE on bud break and RGPin Engelmann spruce seedlings at 268 and 327 days....11512. Protocol for carbohydrate analyses^ 116ixLIST OF ABBREVIATIONSANOVA^ Analysis of variancecc Cubic centimetreCE^ Carbon dioxide enrichmentDF Degrees of freedomDW^ Dry weightg Gravitational accelerationi.d.^ Inside diameterMOF Ministry of ForestsMS^ Mean squareo.d. Outside diameterP ProbabilityRGP^ Root growth potentialSE Standard error of the meanSS^ Sum of squaresSV Source of variationTNC^ Total non-structural carbohydrates$'0 Per mil or per thousand813c^ Stable isotope abundance parameterACKNOWLEDGEMENTSI wish to express my profound gratitude to mysupervisor, Dr. R.D. Guy for the academic guidance renderedthroughout the course of this study. His academicenthusiasm, tireless guidance, and patience will always beremembered. I am equally deeply indebted to the othermembers of my supervisory committee, Dr.'s D.P. Lavender,and P.A. Jolliffe who did not only review this manuscript,but gave both material and moral support to bring this studyto this stage.Special thanks go to Dr. H.G. Weger for his diligentadvice and guidance on carbohydrate analyses, and Dr. S.NSilim for advice and assistance on various laboratorytechniques and procedures. I am also grateful to all thestaff, graduate students and workers of Ponderosa Annex Bfor all the co-operation extended to me in various ways. Iam particularly grateful to A. Balisky who so kindly proofread this manuscript, and from time to time came to myrescue whenever I ran into computing problems.I wish to thank the International Development ResearchCentre (IDRC, Canada) in co-operation with the ZambianGovernment for the financial support that made this studypossible. I am also grateful to Pacific RegenerationTechnologies Inc. who funded a major part of my thesisresearch.I would also like to thank the Zambian community inVancouver and the African students at UBC for the kindness,understanding, and co-operation that I shared with them.Finally, I wish to extend my sincere thanks to my wife,Cecilia, and my two sons, Ian Musakanya and Chama Kizito fortheir patience, perseverance, and encouragement during myacademic struggle. I miss them a million times.xiThis thesis is dedicated to my second bornson, CHAMA KIZITO CHOMBA whom I leftwhen he most needed me.xii11.0 INTRODUCTION.Rapid initiation of adequate and vigorous root growthis necessary for early survival and establishment ofoutplanted seedlings (Burdett 1987, Burr 1989, Ritchie etal. 1985, Sutton 1987). Root initiation and early growthwill depend on environmental factors (e.g. soil moisture,soil temperature, nutrients, photoperiod, etc.), theseedlings' physiological status (e.g. cold hardiness, buddormancy, stress resistance, carbohydrate reserves, etc.),and management practices (e.g. lifting date, freezer-storageduration, site preparation, etc.). This study attempts onlyto examine the role of carbohydrate reserves in spring rootgrowth of freezer-stored Engelmann spruce (Picea engelmanniiParry) seedlings. Emphasis is placed on investigating theutility and possible side effects of carbon dioxideenrichment (CE) on the manipulation of these reserves.During the past twenty years, the reforestation programin British Columbia has been characterized by the plantingof nursery produced seedlings. Prior to 1975, most plantingtook place in the coastal areas of British Columbia. Morerecently, planting efforts in the interior of the provincehave become more important and the species currentlypredominant in the reforestation program include whitespruce (Picea glauca (Moench.) Voss), Engelmann spruce, andlodgepole pine (Pinus contorta Loud.). Furthermore, themajority of the seedlings planted in the interior are, infact, produced at the southern latitudes and lower2elevations. For example, during the 1987 planting season,interior spruce (white spruce and Engelmann spruce, and arehybrids) and lodgepole pine together made up well over 75per cent of the approximately 230 million containerseedlings planted in British Columbia (B.C. MOF 1989,Lavender unpublished data). For the 1990 planting seasononly 208 million seedlings were planted, and the proportionof interior spruce was comparable to the figures of theprevious years. This shift in the emphasis of thereforestation program has necessitated the development ofcold/frozen storage of fall or early-winter liftedseedlings. In cold-storage, the seedling's dormancy andresistance to stress may be maintained until planting timein May/June. If the seedlings are not kept cold, they willbreak their buds in spring in response to the risingtemperature, before the snow melts in the interior. Thecurrent practice is to cold/freeze store interior spruceseedlings four to eight months, prior to outplanting them inspring or early summer.Lack of root growth by outplanted interior spruceseedlings has been noted as one of the major factorscontributing to plantation failure in north-central BritishColumbia, where several hundred thousands of hectares arenot satisfactorily restocked (Butt 1986). This lack of rootdevelopment may have a direct bearing on the reduction ofthe ability of outplanted seedlings to take up necessarymoisture from the soil, and is presumed to cause "planting3check" (Grossnickle 1988). Although root growth may becorrelated with high survival and growth of outplantedseedlings (Burdett 1979, Stone 1955), there seems to be noadequate data to support and explain how the cause-effectrelationship is mediated (Binder et al. 1988, Lavender1988).Initial survival and early growth of outplantedseedlings may largely depend on the physiological readinessof the seedlings to rapidly regenerate new roots in order toestablish intimate contact with the soil matrix, therebybeing able to resume water and nutrient uptake. The abilityof seedlings to promptly and abundantly initiate and/orelongate new roots after outplanting in a favourableenvironment has been referred to by many authors as rootgrowth potential (RGP) or root growth capacity (RGC) (Clearyet a/. 1978, Marshall 1985, Ritchie 1982, 1984, Stone 1955).However, the relationships between RGP and the seedling'sfield performance have not been adequately researched(Binder et al. 1988, Lavender 1988, Ritchie and Tanaka1990), and also not all tests show positive relationships(Binder et a/. 1988, Landis and Skakel 1988). It istherefore important to note that RGP is only a measure orgood predictor of overall seedling vigour (Lavender 1988,Ritchie and Dunlop 1980, Stone 1955). As such, anyinterpretation of RGP test results for predictingoutplanting field performance must consider other factorssuch as site conditions and planting quality (Binder et a/.41988). Furthermore, RGP is not a physiological process perse; it is an integrated manifestation of many importantphysiological processes (e.g. cold hardiness, stressresistance, carbohydrate status, etc.). For this reason RGPhas become a popular and useful indicator of seedling vigorand stress resistance; the rationale being that, if there isany problem with seedling physiology, it should show up as adecrease in the seedling's ability to produce new roots(Lavender 1988, Ritchie 1987).2.0 LITERATURE REVIEW2.1 Carbohydrate Reserves and Root GrowthRoot growth can only proceed at the expense ofavailable metabolic substrates, primarily carbohydrates. Inthe case of conifers, lipids and proteins are also importantreserve materials for root growth. Carbohydrate reservesplay an important role in maintaining respiration and growthduring times when photosynthesis is not taking place (Duryeaand McClain 1984, Kramer and Kozlowski 1979, Ritchie 1984).According to Ritchie and Dunlop (1980) and Duryea andMcClain (1984), the most abundant translocatablecarbohydrate in trees is sucrose. On the other hand, starchis the most abundant storage form of carbohydrate.Furthermore, plants are capable of accumulating and storingcarbohydrates in roots, stems, and foliage for subsequentmetabolism either within the site of assimilation andstorage or for export to other growing tissues (Glerum51980a, Kramer and Kozlowski 1979).In terms of root development, it is not well knownwhether root growth at planting time proceeds at the expenseof stored carbohydrates, at the expense of recently photo-assimilated carbohydrates, or both. Several studies on therole of carbohydrate reserves in root growth potential havepresented conflicting results. For example, Ritchie andDunlop (1980) concluded that initiation of new roots inmost conifer seedlings depends on substances (presumablycurrent photosynthate and growth hormones) originating inthe shoots and translocated through the phloem to the rootsystem. In addition, many studies on root development inDouglas-fir (Pseudotsuga menziesii (Mirb.) Franco) haveshown that the main source of stimulus for spring rootgrowth is the foliage (Philipson 1988, Ritchie 1982, van denDriessche 1987). Results from bark-ringing experiments byZaerr and Lavender (1974) indicated that food reserves inroots of the girdled Douglas-fir seedlings declined steadilyduring the five weeks of the experiment but root activitydropped to zero within three weeks. Results from the aboveexperiment led Zaerr and Lavender (1974) to conclude thatroot activity may require carbohydrates but the level offood reserves alone does not control root growth. Similarly,results from radioisotope experiments with Douglas-fir andSitka spruce (Picea sitchensis (Bong.) Carr), and low CO2concentration and girdling experiments with Douglas-firseedlings, led van den Driessche (1987, 1991) to speculate6that current photosynthate is the primary carbon source forearly root growth. Other researchers have found thatphytohormones may also significantly influence early rootgrowth. For example, Kramer and Kozlowski (1979) statedthat, as dormancy intensity weakens in spring, buds exportincreasing amounts of auxins and gibberellins, which inconifers may move to leaves and stimulate the production oraccumulation of root promoters. In deciduous species whereno leaves are present, rooting promoters remain in the budsor stems (Carlson and Larson 1977). In addition, in bark-ringing experiments, Zaerr and Lavender (unpublished data)demonstrated that some substance (most likely plant growthregulators) besides current photosynthate controlled rootgrowth because 14C-sucrose was fed to the lower edge of thegirdle, roots did not grow, but radioactivity was isolatedin the root tips. In both conifers and hardwoods, growthhormones move downward through the phloem parenchyma toinitiate root growth (Salisbury and Ross 1985). Work onponderosa pine (Zaerr 1967), Douglas-fir (Deyoe and Zaerr1976), and red oak (Quercus rubra L.) (Carlson and Larson1977) seedlings also confirmed that plant growth regulators(particularly auxins and gibberellins) enhanced rootinitiation. In nature, mycorrhizae are also important toroot growth of higher plants by way of increasing theefficiency of nutrient and water uptake, and may affectplant growth hormone physiology (Harley and Smith 1983,Mikola 1980).7On the other hand, other researchers claim that newroot growth largely depends on carbohydrate reserves. Forinstance, Ronco (1973) found that first year survival ofoutplanted Engelmann spruce seedlings seemed to depend onfood reserves accumulated while in the nursery. In addition,Kozlowski and Keller (1966), and Krueger (1967) speculatedthat since the rate of carbon assimilation of newly plantedseedlings is rather low until the time they becomeestablished, it is logical to assume that new root growthdepends on stored carbohydrates. Marshall (1985) expressedthe view that seedlings are dependent on reservecarbohydrates from the time they are lifted untilphotosynthesis is sufficient to meet the demands of growthand respiration. He further pointed out that if carbohydratereserves are inadequate to meet the respiratory demandsassociated with the cold storing and outplanting of theseedlings, the seedlings will die. In a bark-ringingexperiment by Philipson (1988), root growth in Sitka spruceseedlings showed dependance on carbohydrate reserves, whileroot growth in Douglas-fir seedlings depended entirely onrecently assimilated photosynthate. Contrary to the abovefor Douglas-fir, findings in support of carbohydratereserves being responsible for early root growth followingstorage were reported by Krueger and Trappe (1967). Based onthe available literature, it appears that the resultsobtained so far have mostly been with coastal species,which, in terms of overwintering strategy, may be quitedifferent from interior species.2.2 Carbohydrate Reserves and CO2 EnrichmentOne way to assess the role of carbohydrate reserves inRGP is by producing seedlings with different levels ofstarch reserves prior to placing them in cold-storage. Onepossible way to increase carbohydrate reserves is throughCO2 enrichment of the seedlings before taking them intocold-storage. The positive effects of short-term CO2enrichment on the growth and yield of plants have beenwidely demonstrated (Campagna and Margolis 1989, Gifford1979, Jolliffe and Ehret 1984, Mortensen 1987, and Waggoner1984). According to Mortensen (1987), the optimal CO2concentration for plant growth and yield ranges from 700 to900 gL•L-1 CO2 . This is approximately two to three timeshigher than the current atmospheric CO2 concentration (ca.340 gL•L -1 CO2).However, the responses of plants to increasing CO2concentration will mostly depend on environmental factorssuch as soil moisture, nutrients (particularly nitrogen andphosphorus), soil temperature, and light intensity. Forexample, Brown and Higginbotham (1986) found that CO2enrichment significantly increased total dry weight of whitespruce and aspen (Populus tremuloides Michx) seedlings grownin a high nitrogen regime as opposed to a low nitrogenregime. Conroy et al. (1990) also indicated that, both highphosphorus and CO 2 levels increased the total dry weights89and growth rates of Pinus taeda D. Don and Pinus caribaeavar. hondurensis seedlings, and that these effects weresynergistic. Other researchers found positive andsynergistic effects of CO2 enrichment and high irradiance onthe growth rates and biomass production of seedlings (Tolleyand Strain 1984, Yeatman 1970). For instance, Tolley andStrain (1984) reported that elevated CO 2 concentration (ca.650 gL•L -1 CO2 ) and high irradiance (ca. 1000 gmol quantam-2s-1 ) increased total dry weight of sweetgum (Liquidambarstyraciflua L.) and Pinus taeda L. seedlings. In bothspecies, these effects were highly dependent on age (theyounger the seedlings the more responsive they were), andduration of exposure (prolonged exposure resulted in notreatment effects). It is also important to note that theeffects of elevated CO2 concentration are species specific,with some species responding more positively than others(Brown and Higginbotham 1986, Kramer 1981, Sionit et al.1985, Tolley and Strain 1984).Results from CE experiments with black spruce (Piceamariana Mill.) by Campagna and Margolis (1989) showed nosignificant differences in the concentrations of sugars,starch, or total nonstructural carbohydrates (TNC) in eitherroots or stems after three to six weeks exposure to elevatedCO2 levels. On the other hand, CE significantly increasedstarch concentrations in the needles. Furthermore, Yeatman(1970) demonstrated that elevated CO2 concentrations withhigh irradiance increased the seedling dry weight of species1 0such as white spruce, Norway spruce (Picea abies (L.)Karst), jack pine (Pinus banksiana Lamb.), and Scots pine(Pinus sylvestris L.), by 30 to 80 per cent at 1,000 µL•L -1CO2. Campagna and Margolis (1989), and Surano et al. (1986)also found similar responses with black spruce and ponderosapine (Pinus ponderosa Dougl. ex P. Laws.) seedlingsrespectively. However, very little is known about the effectof CO 2 enrichment on starch concentration in coniferseedlings. The optimum stage at which CO2 enrichment willeffectively increase carbohydrate reserves during seedlingproduction in the nursery is also not known. For example,virtually none of the early work on CE has examined theeffects of CE on carbohydrate reserves after bud set, whenheight growth ceases but photosynthesis continues. Moreover,according to the views of many researchers (Hollinger 1987,Kramer 1981, Surano et al. 1986), in forestry, the long termeffect of elevated CO2 concentrations on the growth andphysiological conditions of seedlings has received toolittle attention. The present study attempts to examine theeffects of CE on carbohydrate reserves prior to cold-storage, and on post-storage RGP in Engelmann spruceseedlings.2.3 Carbohydrate Reserves and RGP During StorageDuration of cold-storage has been implicated in theappearance of adverse effects on the physiological conditionof seedlings. For instance, Loescher et a/. (1990),1 1McCracken (1979), Ritchie (1982), and van den Driessche(1979) stated that one important change occurring in cold-stored seedlings, which might account for changes inperformance, is the gradual respiratory depletion of reservesugar and starch. This stems from the fact that storedseedlings,^which are no longer able to activelyphotosynthesize,^slowly metabolize their carbohydratereserves. Loss of such reserves has been implicated as beingresponsible for poor survival and poor RGP (Hellmers 1962,Stone 1955, 1970, Stone and Jenkinsen 1970). Carbohydratedepletion in stored seedlings has been extensively studiedin Douglas-fir by Ritchie (1982), in jack pine by Glerum(1980b), in ponderosa pine by Hellmers (1962), in Engelmannspruce by Ronco (1972, 1973), and in mugo pine (Pinus mugoTurra var. mughus) and in radiata pine (Pinus radiata D.Don) by McCracken (1979). All these authors hypothesizedthat poor seedling performance at planting time wascorrelated with a depletion of carbohydrate reserves duringcold-storage. For interior spruce seedlings, Ritchie (1985,1987) and Ritchie et a/. (1985), indicated that RGP isrelated to dormancy release. Generally speaking, RGPincreases as buds accumulate chilling hours and finallypeaks when the chilling requirement has been fulfilled.Thereafter, it declines rapidly, as the internal metabolicfocus of the seedlings switches to the elongation of the newshoots (Ritchie and Dunlop 1980, Ritchie and Tanaka 1990).122.4 Stable Carbon Isotopes as TracersAnother approach to resolving the carbohydrate reservecontroversy and establishing the absolute contributions ofold and new photosynthate to post-storage root growth, is bythe use of stable isotopes. Until recently, most C-labellingexperiments have employed radioactive 14C (van den Driessche1987, Glerum 1980a, Ursino et a1. 1968), but regulatoryconstraints associated with use of radioactive materialsmake some such studies awkward, particularly long-termlabelling experiments and field studies (Svejcar et al.1990). In addition to regulatory constraints, the period forradioactive 14C labelling is short which may result ininsufficient or uneven labelling of plant material. On theother hand, because of the advantages of safety, longerperiod of labelling, and increased availability of isotoperatio mass spectrometers, stable carbon isotope techniquesare beginning to gain in popularity for use in tracerstudies in plants (Svejcar et al. 1990, Mordacq et a/. 1986,O'Leary 1981). As used in this study, the technique involvesgrowing seedlings, prior to storage, in air containing CO2with different proportions of 13C and 12C. Later, thechanges in the 13 C/ 12C ratio can be determined in the newroots produced after the storage period (Glerum 1980a,Svejcar et al. 1990). If after cold-storage such seedlingsare allowed to grow under normal air, all new photosynthatewill contain a different 13 C/ 12C ratio. Stable isotopelabelling approaches such as this, particularly at thenatural abundance level, have not been extensively used instudies of plant/tree physiology and the present study issomewhat unique in trying to explore these possibilities.2.5 Soil Temperature and Root GrowthRegardless of the condition of the seedlings, soiltemperature may exert a major influence on root developmentand ultimately establishment. Studies by Heninger and White(1974), and Hermann (1962) revealed that expression of RGPin transplanted seedlings was highly dependent on soiltemperature. Camm and Harper (1991) also found that rootgrowth of over-wintered, cold-stored white spruce seedlingswas temperature dependent. However, it is interesting tonote that the effect of soil temperature on root developmentappears to be species specific such that different specieshave different optimal soil temperatures. As noted byRitchie and Dunlop (1980), and Lavender and Overton (1972),root development proceeds favourably between 18 and 25 °C,depending upon the species, with those species native tocooler climates tending to have a lower optimal soiltemperature (e.g. interior spruce: 19 °C) than those nativeto warmer climates (e.g. loblolly pine: 25 °C). On the otherhand, Husted and Lavender (1989) reported relatively loweroptimal soil temperature for white spruce (e.g. 15-17 °C)than the figures quoted above for interior spruce.In the interior, soil temperatures at planting time aregenerally far below the above mentioned figures. The effect1314of such low soil temperatures on plantation establishment isthrough a slowing of early root development and shoot growth(DeLucia 1986, Grossnickle 1988, Grossnickle and Blake1985), presumably due to decreased metabolic activity and adecreased turgor of root cells caused by reduced wateruptake (Lopushinsky and Kaufmann 1984). According toLopushinsky and Kaufmann (1984), the main causes ofdecreased uptake of water at low soil temperatures areincreased viscosity of water and decreased permeability ofroot cell membranes. Furthermore, there are speculationsthat carbohydrate reserves play an important role in osmoticadjustment for purposes of increasing water uptake inconifer roots growing at low temperatures (Ritchie 1982,DeLucia 1986). The occurrence of high carbohydrate levels inroots of seedlings grown at low soil temperature as opposedto high soil temperature, may in part, reflect this role.For example, Weger and Guy (1991) demonstrated that bothsoluble sugar and starch levels in white spruce wereapproximately 50% higher in 4 °C-grown roots than 11 and18 °C-grown roots. Marshall and Waring (1985) reportedsimilar trends in starch and soluble sugar content in theroots of Douglas-fir seedlings grown at 10, 20, or 30 °C soiltemperatures. On the other hand, experiments with Douglas-fir (Ritchie 1982) and Engelmann spruce (DeLucia 1986)seedlings revealed that conversion of starch to sugars inroots is an important acclimation response to lowtemperatures. The rationale being that, sugars depress the15freezing point of the cell sap, tend to concentrate in thevacuole and reduce the probability of intracellular icecrystal formation, and may serve some cryoprotective rolewith respect to cell membranes (Santarius 1982). For thisreason, it is probably right to assume that carbohydratesplay an important role in frost hardiness. As noted by manyauthors (Guy 1989, Levitt 1980, Santarius 1982),accumulation of free sugars in both woody and perennialplants seems to be a requirement for cold acclimation.2.6 OBJECTIVESBased on the foregoing literature review, this studywas conducted with the following objectives in mind:1. To investigate the effects of CE on carbohydrate levelsand post-storage root growth in Engelmann spruce seedlings.In addition, the effects of CE on other morphological andphysiological characters were examined.2. To observe changes in carbohydrate levels during cold-storage.3. To measure relative proportions of old carbon (fromstored carbohydrates) and new carbon (from currentphotosynthate) in new roots of cold-stored Engelmann spruceseedlings.3.0 MATERIALS AND METHODSThe study consisted of two experiments. The firstexperiment involved producing seedlings under six CO2regimes and then storing them during winter for twodifferent storage durations. This was followed by plantingthe seedlings in growth chambers at 268 and 327 days (i.e.after two and four months of storage, respectively). Thesecond experiment involved producing seedlings in thenursery using the standard nursery practices up to bud set(September). Thereafter, seedlings were differentiallylabelled with 13CO2 until the seedlings were ready for cold-storage.3.1 Experiment One: CO2 Enrichment and Carbohydrates3.1.1 Seedling Production and Cold-storageEngelmann spruce seedlot number 8356^(location:Spapilum, 51 °14' latitude, 119 °39' longitude) was sown inMay, 1989 in the Plant Science Laboratory at UBC. The seedswere sown in eight home-made growth cabinets belonging toDr. Jolliffe, Plant Science Department, and CO2 enrichmentcommenced immediately. The CO 2 regimes consisted of high andlow concentrations (i.e. 1000 gli•L -1 and 340 1.1L•L -1 of CO2respectively), and reciprocal transfers of seedlings fromlow to high and high to low CO2 concentrations. Transferswere conducted after 60 and 120 days of CO2 treatment. Airwas pulled into the chamber from outside the MacMillanbuilding by fans mounted in the base of each cabinet (Fig.16171). Two chambers were used and each one of them was dividedinto 4 compartments, with replicate treatments arrangedFigure 1. A general view of the CE experiment during the seedlingproduction phase.18diagonally opposite each other. Tank CO2, regulated manuallyby adjusting rotometer valves, was added to the incoming airfor the high CO 2 cabinets. Carbon dioxide concentrations inall the cabinets were monitored with an infrared gasanalyzer (IRGA, LI-864 CO2 Analyzer from Beckman, Fullerton,CA). Air temperature and humidity in each chamber wererecorded continuously with a Campbell Scientific (Logan UT)21X data logger. On average the growth cabinet environmentwas as follows: day and night temperatures 22/15 °C, 18 hourphotoperiod initially and then reduced to 9 hours at 100days to induce bud set, which followed within 2-3 weeks,photosynthetic photon flux density ca. 190 Rmol quanta m-2 s -1 (full sunlight is about 2000 Rmol quanta m -2 s -1 ), andrelative humidity 70-75% during the day and 85-90% at night.Initially, the root medium contained slow releasefertilizer, but seedlings were also fertilized on a regularbasis with 20:8:20 N:P:K and micronutrients. Seedlings weregrown for 180 days following emergence, then hardened off.Seedlings were hardened off by moving them into water-cooledPlexiglas fumigation boxes within a Conviron E15 (Winnipeg,Manitoba) growth chamber. Day/night temperatures werereduced to 8/5 °C. Other conditions remained the same and theCO2 treatments were continued for three weeks, by which timecold hardiness had reached at least -22 °C (Silim: personalcommunication). Thereafter, seedlings were packed and thentaken into cold-storage (at -5 °C) for two and four months.The first transfer was done at 60 days and the second19one at 120 days. At 80, 120, 140, and 202 days, someseedlings were randomly sampled from each treatment. Thesampled seedlings were killed in liquid nitrogen, freeze-dried, and then they were kept in a freezer for latercarbohydrate analysis. At each harvest date, growth datawere also collected. At 202 days, the remaining seedlingswere sorted according to treatment, packed and taken intocold-storage (-5 °C). There were other harvests prior to 80days, but these are not included in the thesis becauseemphasis was placed on the examination of CE effects aftertransfers (i.e. 60 and 120 days) and bud set.3.1.2. Post Cold-storage PlantingAt 268 and 327 days, seedlings were removed from cold-storage and allowed to thaw for seven days (at 5 °C).Seedlings were then randomly selected, potted, and placed inthree water baths (3 °C, 7 °C, 11 °C for 268 days; and all at11 °C for 327 days) in a Conviron E30 (Winnipeg, Manitoba)growth chamber at the UBC South Campus Nursery. Theintention was to have another planting at 388 days (i.e.after 6 months storage), but the seedlings scheduled forthis planting date were spoiled when the cold-room failed.For the root medium, peat/perlite (2:1) was used withdolomite lime added to the mixture to adjust the pH. Toraise the root medium pH of approximately 4.65 to about 5.5,600 g of dolomite lime was added to 20 L of peat/perlitemixture. Silica sand (800 mL) was placed at the bottom of20each pot to keep the pots in place.There were nine pots per water bath (all six CO2treatments represented) with four seedlings per pot (Fig.2). Styrofoam covers were installed over each water bath tominimize heat transfer to and from the air around pots. Thestyrofoam covers also facilitated keeping the pots in place.A thermometer was placed in each water bath to monitor theroot zone temperature. Another thermometer was suspendedabove the seedlings in each water bath to cross-check theroom air temperature with the temperature set on the growthchamber control system. No fertilizer was added to eitherthe root medium or the water, because 28 days growing periodin the growth chamber was considered to be relatively shortfor the seedlings to become nutrient deficient. At plantingtime seedlings were watered to field capacity (900 mL ofwater/pot). Thereafter, seedlings were watered every 3-4days (50 mL/pot) or whenever necessary to maintain the rootmedium at field capacity.Growth chamber conditions during the post-storageperiod were as follows: temperature 11 °C day and night,relative humidity 70%, photoperiod 16 hours, photosyntheticphoton flux density 350 gmol quanta m -2 s -1 , from cool whitefluorescent lights (Philips VHO), supplemented withincandescent bulbs. Seedlings were grown for 28 days duringwhich time bud break assessments were conducted every twodays. On the 28th day, seedlings were removed from thegrowth chamber, their root medium carefully washed off, andnew white roots counted (i.e. new roots > 5 mm long).21Figure 2. A general view of the pot arrangement of the CEexperiment in the growth chamber in one of the water baths.3.1.3. Carbohydrate AnalysesAt each planting time, some seedlings were randomlyselected (all six CO2 treatments represented) and harvestedfor analysis of starch and soluble sugar content. Theharvested seedlings were divided at the root collar intoroots and shoots, killed by freezing them in liquid nitrogenand then they were freeze-dried for 3-4 days. Together withthe previously harvested samples, starch, soluble sugars,and their sum, total non-structural carbohydrates (TNC),were determined by methods modified from da Silveira et a/.(1978), Haissig and Dickson (1979), Jermyn (1975), Rose etal. (1991), and Yemm and Willis (1954). Seedlings from thefirst set of reciprocal transfer experiments (60 days aftersowing) were included in the analysis only at 80 and 120days. For the entire experiment, a total of 1284carbohydrate samples were analyzed.3.1.3.1. Soluble Sugars Analysis by Anthrone ReagentSoluble sugars (i.e. free sugars), expressed as glucoseequivalents, were determined by methods modified from Jermyn(1975), and Yemm and Willis (1954). Details of theanalytical procedure are reported in Appendix 12 (item V).The already freeze-dried plant material (i.e. needles,stems, and roots) was finely ground in liquid nitrogen witha mortar and pestle. Twenty-mg samples were extracted withfive mL methanol:chloroform:water (M:C:W) (12:5:3, v/v/v)overnight. After centrifugation (desk top) the pellet was2223re-extracted with three mL M:C:W. Supernatants from the twoextractions were then combined, and three mL of distilledwater were added. After centrifugation, the aqueous phasewas removed and evaporated to dryness. Thereafter, the driedsample was dissolved in 3 mL of distilled water and storedfrozen until required for sugar analysis. Prior to analysis,200 gL samples were treated with 50 gL each of Ba(OH)2 andZnSO4 (0.3N) to remove organic acids and proteins, andsoluble sugars were analyzed by anthrone according to Jermyn(1975), and Yemm and Willis (1954).3.1.3.2. Starch Analysis by Enzymatic HydrolysisStarch was analyzed by gelatinizing the pellet fromthe two extractions above in five mL of acetate buffer(150mM, pH 4.5) and autoclaving at 120 °C for one hour. Afterthe mixture was cooled to 55 °C in a water bath, 125 units ofamyloglucosidase (E.C. 3.2.1.3; from Rhizopus, Sigma A-7255)and 25 units of a—amylase E.C. 3.2.1.1 (from Aspergillus,Sigma A-0273) were added to the samples, and the mixtureswere then incubated for two hours. Thereafter, the mixtureswere centrifuged (desk top), and the supernatants analyzedfor starch (expressed as glucose equivalents) using glucoseoxidase-peroxidase-o-dianisidine (Ebell 1969, Haissig andDickson 1979, Rose et a/. 1991). For more details on theanalytical procedures, refer to Appendix 12 (item IV).243.2. Experiment Two: Stable Carbon Isotopes as Tracers3.2.1. Seedling Production and Cold-storageThe second experiment involved producing Engelmannspruce seedlings (the same seediot as in the firstexperiment) in 213 styrofoam blocks at the UBC South CampusNursery beginning in May, 1990. The standard nurseryprocedures were followed up to bud set (September).Thereafter, seedlings were moved into a Conviron E30 growthchamber, and placed in four water-cooled 125 dm 3 Plexiglasboxes for stable carbon isotope "labelling" (Fig. 3). Eachbox contained 50 seedlings; two boxes acted as controls(i.e. receiving ambient CO2 with a normal isotopiccomposition) and the other two were "labelled" (i.e.receiving air that was first stripped of ambient CO2, beforeadding back CO2 depleted in 13C). A computer dataacquisition and control system (WB -820 Omega Engineering,Inc., Stamford, CT) and mass flow controllers (MFC 825,Edwards High Vacuum Intl, Wilmington, MA) matched CO2concentrations in "labelled" boxes with the "unlabelled"boxes by injecting tank CO2 into the former. The IRGA (LI-6251 CO2 Analyzer from LI-COR, Lincoln, NB) was used tomonitor CO2 concentrations in the boxes. The Plexiglas boxeswere provided with small fans and de-humidifying airrecirculation loops. Tank CO2 going into "labelled" boxeshad a 513C value of -35.81%.Initial growth chamber conditions were as follows: dayand night temperatures 22/15 °C, photoperiod 12 hours,25Figure 3. A schematic diagram of equipment used to produceseedlings differing in stable carbon isotope composition(isotope labelling experiment). Two of four boxes are shown.Air taken from outside was split into two streams, and onestream was pumped directly into control (i.e. "unlabelled")boxes. The second stream was stripped of CO2 by passingthrough soda lime before being pumped into boxes for the"labelled" seedlings. An infra-red gas analyzer (IRGA) wasused to monitor CO2 concentrations. A computer dataacquisition and control system and mass flow controllers(MFC) matched CO2 concentrations in the "labelled" boxeswith the "unlabelled" boxes by injecting tank CO2 into theformer. The computer also monitored and controlled the airtemperature and humidity in the boxes.Air OUTTank CO2(5 13C = —36 7«,26"unlabelled"Pump ^ Air OUTOutside Air IN= —8 %.IRGA!44 1't I "labelled"MFC■••■•■•■■ SodaLime.■•■11. Filter Pump•■■•=01•427relative humidity 50% during the day and 60% at night, andphotosynthetic photon flux density ca. 300 lamol quanta m-2s -1 . On November 30, 1990, the temperature in the growthchamber was reduced to 10/5 °C day/night in order to harden-off the seedlings before putting them into cold-storage.Seedlings were watered to field capacity every three days(i.e. 2.5 L of water per box, allowed to freely drain for 10minutes). Furthermore, 20:8:20 N:P:K fertilizer was appliedto the seedlings once a week for the first four weeks andthen once every fortnight thereafter.On December 19, 1990, seedlings were lifted, packed inplastic-wrap, placed in lined boxes and taken into cold-storage. At the same time, six seedlings were harvested fromeach treatment (i.e. control and labelled). The harvestedseedlings were then killed in liquid nitrogen, freeze-dried,and stored at -5 °C until required for carbohydrate andstable carbon isotope analyses.3.2.2. Post Cold-storage PlantingFor the carbon isotope tracer study, seedlings werewithdrawn from the cold-storage for planting in the growthchamber at the UBC South Campus Nursery only once, in April,1991. Growth chamber conditions and root medium were asdescribed in section 3.1.2. However, here 30 pots wereplaced in each water bath (three water baths all at 11 °C)and only one seedling was placed in each pot. Hence, therewere 15 pots for the "labelled" seedlings and 15 pots forcontrol seedlings, all randomly placed in each water bath(Fig. 4).(A)(B)Figure 4. General views of the equipment arrangement whenseedlings were moved into the Plexiglas boxes for (a)isotope labelling, and (b) during post-storage planting.2829Also at planting, six seedlings from each treatment wereharvested and preserved for carbohydrate and isotopeanalyses.Seedlings were grown in the growth chamber for 36 days.On May 9, 18, and June 5, 1991, five seedlings for eachtreatment were harvested from each water bath. The rootmedium was carefully washed off the harvested seedlings, newwhite roots collected, killed in liquid nitrogen, freeze-dried, and then stored in the freezer until required forstable carbon isotope analysis.3.2.3. Stable Carbon Isotope AnalysesBefore the new roots were analyzed for 13 C/ 12 C ratio,the already harvested pre- and post-cold-storage sampleswere analyzed for carbohydrate reserves. The analyticalprocedures in chapters 3.1.3.1 and 3.1.3.2 were followed.The carbohydrate analysis at this stage was necessary onlyfor establishing the level of carbohydrate reserves in theplant material we were dealing with. In addition, before theactual 13 C/ 12C ratio analysis, samples (new white roots,starch and soluble sugars extracts) were prepared. Thesamples were finally assessed for 13 C/ 12C ratio using themethods adapted from Boutton (1991), Deleens et al. (1989),Engel and Maynard (1989), Guy and Wample (1984), and Svejcaret al. (1990). In essence the methods involve changing theplant material into CO2 by combustion for determination ofthe 13 C/ 12C ratio by mass spectrometry. The isotope analysis30was performed by the Isotope Mass Spectrometry Laboratory,Department of Oceanography, using a VG Isogas Prism triple-collecting mass spectrometer. The 813C was calculated fromthe measured carbon isotope ratios of the sample andstandard gases as:813CW = [(Rsample-Rstandard)/Rstandard] x 10 3where 813C is the parts per thousand, or per mil (tD),difference between 13C content of the sample and thestandard, and R is the mass 45/44 ratio of sample orstandard gas. The 8 13C values were expressed relative to theinternational PDB (Pee Dee Belemnite) standard. Samplepreparation and machine precision was ± 0.1% -o. The fractionalabundance (F) was estimated by the following equation:F^(RL-RC)/(CL-CC)where F is fraction of old carbon, RL is current labelledroot 813C, RC is current control root 813C value, CL islabelled old carbon source (i.e. carbohydrate) 5 13C, and CCis control old carbon source (i.e. carbohydrate) 8 13C value.The values used for C L and CC are weighted means of separate813C values for sugars and starch.313.2.3.1. New White RootsDue to the small amounts of tissue involved, thefreeze-dried root samples were not ground or pulverized in aconventional way (i.e. grinding with a mortar and pestle orin a Wiley mill). Instead, these were ground as finely aspossible within their storage vials using a clean glass rod.The ground samples were kept in a desiccator under vacuumuntil required for combustion.Combustion tubes were prepared from quartz tubing (6 mmo.d. x 4 mm i.d.) cut into 25 cm lengths, fused shut at oneend. Tubes were loaded with ca. 1.55 g of cupric oxide wire(pre-fired at 550 °C), 2.0 g reduced copper and plant sample(3 - 10 mg). The sample tubes were then attached to a vacuumline, evacuated to less than 0.13 Pa (10 -3 torr), and sealedwith a torch. Sealed sample tubes were placed in a mufflefurnace and combusted at 900 °C for two hours to convert allthe organic carbon to CO2 quantitatively. The combustedsample tubes were stored at room temperature for a month,but were reheated to 550 °C for ca. one hour just prior toisotope analysis. Reheating of samples was necessary becauseEngel and Maynard (1989) found that, if combusted tubes arestored for more than five days before analysis, CO2 becomesdepleted by 1-3% as a result of carbonate formation. Ifsamples cannot be processed within five days, this isotopicdepletion can be overcome by recombusting the tubes prior tothe actual isotope analysis (Engel and Maynard, 1989).323.2.3.2. Starch ExtractionSix 100 mg samples of the already ground plant materialfrom each treatment were pooled together, representing four600 mg samples (i.e. two for pre- and post-cold-storagetreatments respectively). Separation of soluble sugars fromstarch and the preliminary extraction of soluble sugars weredone following the procedures outlined in Appendix 12 (itemsII and III). However, in this case, volumes for M:C:W anddistilled water were increased four-fold to account for theincreased amount of plant material. The starch pelletrecovered was then freeze-dried, and starch was finallyextracted by methods adapted from Brugnoli et al. (1988),Ehleringer (1991), and Hassid and Abraham (1957).Two hundred and fifty mg of the starch pellet wasweighed out and placed in a 25-mL centrifuge tube. Then 25mL of boiling 80% ethanol were added and the mixture boiledin a water bath for 15 minutes. After cooling, the sampleswere centrifuged (desk top) and the supernatant discarded.The extraction was repeated until the supernatant wascolourless. The pellet was re-suspended in 20 mL of 20%hydrochloric acid (w/w) for 30 minutes, spun down (desk top)and the supernatant saved. The pellet was re-extracted with20 mL of 20% hydrochloric acid and spun down as before. Thetwo supernatants were then combined in a volumetric flask,made up to 100 mL with distilled water, and then 6.6 g ofsodium chloride and 10 mL of 0.14N iodine-potassium iodidesolution were added and left standing on ice for 45 minutes.33Thereafter, the precipitate was centrifuged in 250 mLcentrifuge bottles at 1000 g for 10 minutes at 5°C (BeckmanJA-14 rotor). The supernatant was discarded. The pellet wasthen re-suspended in alcoholic sodium chloride (0.34N in 70%ethanol) (4x5 mL) for transfer to a 25 mL centrifuge tube,spun down (desk top), and the supernatant discarded. Four mLof 0.25N alcoholic sodium hydroxide was added to the pelletand left in the refrigerator overnight (repeated wherenecessary until the brownish colour disappeared). Thefollowing day, the alcoholic sodium hydroxide was pouredoff, the starch pellet was re-suspended (2x) in 15 mL of60% ethanol, centrifuged (desk top), and the pellet saved.The starch pellet was then dissolved in 10 mL ofdistilled water by autoclaving at 120 °C for one hour. Thealiquot of starch extract was filtered, and thenreprecipitated by adding 15 mL of 100% ethanol. Theprecipitation did not occur spontaneously, and so another 10mL of 100% ethanol were added and the samples were then keptin the freezer for approximately two days. Thereafter, theprecipitate was centrifuged at 12,000 g (Beckman JA-20rotor) for 20 minutes at 5 °C, and the supernatant discarded.Fifteen mL of 60% ethanol were added to the pellet andallowed to sit for five minutes. The fluid was discarded andrinsing was repeated with 95% ethanol, 100% ethanol, andthen ether (2x). The starch extract was finally air-dried atroom temperature for ca. 30 minutes, and the samples storedin a desiccator until needed for isotope analysis.343.2.3.3. Soluble Sugars ExtractionMethods described by Brugnoli et al. (1988) andEhleringer (1991) were adapted to extract soluble sugars.First of all, resins (Dowex-50 (11 + form) and Dowex-1 (C1 -1form)) were prepared as follows: Both resins were washedwith distilled water several times to remove fines and bringthe slurry pH to that of distilled water (ca. 6.0). Foractivation, 10-fold excess of 1N HC1 and 2N HC1 were addedto Dowex-50 and Dowex-1 respectively. These were allowed tostand, with occasional stirring, for 15 minutes. Thereafter,the slurry was filtered, and washed well with distilledwater until the pH was approximately 6.0.Five mL of each resin were loaded into separate 10-ccLuer-Lock syringes. These "columns" were arranged in serieswith the Dowex-1 on top, and the Dowex-50 below. Aqueousfractions obtained from the preliminary soluble sugarsextractions (12 mL per treatment) were then loaded onto thecolumns and eluted with 180 mL of distilled water. Theeluate was then filtered, and flash evaporated to a finalvolume of two mL. The samples were re-filtered, freeze-dried, and then stored in a desiccator under vacuum, untilrequired for isotope analysis.353.3. Experimental Design and Data Analysis3.3.1. Experiment OneThe experimental design was divided into two parts. Thefirst part investigated carbohydrate reserves and seedlinggrowth prior to cold-storage, in which case, a completelyrandomized design was used (Appendices 1-10) Six dates(i.e. at 80, 120, 140, 202, 268, and 327 days) wereconsidered, and at each date only the CO2 treatment effectswere statistically analyzed. Time, as a factor, was notincluded in the analysis. There is ample evidence in theliterature indicating that carbohydrate levels in conifersfluctuate seasonally and overall trends observed in thepresent study were as expected. Four treatments wereconsidered: low, high, step-down, and step-up CO2concentrations. The number of replicates for each treatmentvaried considerably from one month to another; with n=12 for80, 120, and 140 days, n=7 for 202 days, n=2 or 6 (dependingon treatment) for 268 days, and n=6 for 327 days.The second part of Experiment One involved assessmentof bud break and root growth at 268 and 327 days. Theexperimental design at these two dates was similar, exceptfor a slight modification at 327 days. At 268 days acompletely randomized block design with a 2 x 2 factorialarrangement (i.e. six CO2 treatments and three water bathtemperatures) was used (Appendix 11). However, it is worthmentioning here that, since only one water bath was used foreach water bath temperature for the entire growth period,36there was no real replication of the water bathtemperatures. According to Hurlbert (1984), this situationcan be viewed as pseudo-replication. In the context of thisstudy, pseudo-replication may refer to the testing for waterbath temperature effects without true replication of thetreatments. In this study the assumption was made that thegrowth chamber environment was reasonably uniform and thatwater baths differed only with respect to temperature. Thenumber of replicates for each treatment remained fairlyconstant (n=6) from one water bath to another, except for afew situations with n=5 or 7.At 327 days, the same design used for 268 days wasapplied. However, in this case, all the three water bathswere at 11 °C. The number of replicates (n=6) for eachtreatment remained consistent from one water bath toanother.The statistical analysis involved analysis of variance,and the computer statistics program, Systat, version 5.0(Wilkinson 1990) was used to do the analysis. Thesignificance was tested at the P<0.01 level. The 1% level ofsignificance was used in order to account for multiple andpost hoc hypotheses, based on the Bonferroni inequalityprocedure (Meddis 1984, Wilkinson 1990). Group varianceswere tested for homogeneity using Bartlett's test (Walpole1982). Every ANOVA had homogeneous variances. Where theanalysis of variance showed significant differences, meanseparation was accomplished by Tukey's test (Ott 1984, andZar 1984) at the P<0.01 level.3.3.2. Experiment TwoAs in 3.3.1, the experiment was divided into two parts.Part one of the experiment covered pre- and post-coldstorage data for carbohydrate reserves. A completelyrandomized design with a 2 x 2 factorial arrangement (i.e.two storage periods and two treatments: control and"labelled") was used. The control and "labelled" treatmentswere replicated six times (n=6).The second part of Experiment Two covered data for thestable carbon isotope analysis. The initial intention was tohave a completely randomized block design, representing twotreatments and three water baths (all water baths at 11 °C).However, due to low rooting capacity by the seedlings it wasnot possible to perform a two way analysis of variance.Instead the data were subjected to a t-test, with n=9 forthe second harvest, and n=6 for the third harvest. For thefirst harvest only one "labelled" seedling produced some newwhite roots, hence it was not possible to perform anystatistical analysis on a single observation.The computer package used in 3.3.1 for the statisticalanalysis was applied here too. The significance was testedat the P<0.01 level.374.0 RESULTS4.1 Carbon Dioxide Enrichment Experiment4.1.1 Effects of CE on Growth Prior to Cold-storage4.1.1.1 Biomass Accretion and PartitioningBiomass accretion in individual tissues withinseedlings, in response to CE, varied greatly at differentstages of seedling development (Figs. 5 and 6a). From Figs.5 and 6a it can be noted that, CE significantly (P<0.001)affected needle and stem biomass at 80, 140, and 202 days,and root biomass at 80 and 140 days. In all the individualplant parts, there was a general positive response inbiomass production to high CO2 and step-up treatmentsrelative to low CO 2 concentration. The step-down treatmenthad a negative response relative to its control (i.e. thehigh CO2 treatment) but still showed greater final biomassproduction than the continuous low CO2 treatment. Even wherethere were no significant differences between treatmentmeans, high, step-down, and step-up CO2 treatments generallyrecorded higher dry weights than the low CO2 treatment.At the end of the growing season (202 days), needlesrecorded the highest biomass followed by roots, and thestems recorded the least. In all the individual planttissues, the most significant effects of CE were after budset (140 and 202 days). Because after bud set, shoot heightgrowth had almost ceased, increased biomass at this stage ofseedling production could be attributable to increased3839girth, root growth or reserve accumulation (mostly sugarsand starch).Total seedling biomass followed about the same trend asthe individual tissues (Fig. 6b). At all stages of seedlingdevelopment, except at 120 days, CE significantly (P<0.001)affected total seedling biomass. Here also, the pattern ofhigh CO2 levels being superior to low CO2 levels wasprevalent. It is also important to note that, after bud set,both individual tissues and total seedling biomass forenriched seedlings were almost two-fold greater than forseedlings grown at ambient CO2 levels.The shoot:root ratios (on a dry weight basis),decreased steadily during the whole study period and werenot generally affected by CE (Fig. 7). This pattern ofdecreasing shoot:root ratios, especially after bud set wasas expected because at this stage of development the shootsink strength would be minimal owing to cessation of shootheight growth. The significant (P<0.001) effect of CEobserved for the step-down treatment at 120 days stands outbut does not conform to any general pattern, and is withoutobvious explanation.40Figure 5. Effects of CE on (a) needle dry weight, and (b) stemdry weight of Engelmann spruce seedlings. The value for eachbar is a mean of 24 seedlings. Step-down and step-uptreatments represent seedlings that were transfered betweenCO2 environments at 60 and 120 days. Step-up and step-downdata presented for 80 and 120 days are for transfersperformed at 60 days, while further data are for transfersperformed at 120 days. At each age, bars accompanied by thesame letter(s) or no letters were not significantlydifferent at the P<0.01 level.111/ Low CO 211 High CO 2N'S1 Step—downES) Step—upiSEbb11111 Low CO27-1 High CO2^ Step—down22 Step—up±SEb(A)4112001000E800rn600ID^400wco20080^120^140^202Age (days)(B)80^120^140^202Age (days)600500400300200100(B)2500 ^2000CnE1500a)• 10000.00- 500NMI Low CO 2ri High CO 217KI Step-downEgl Step-up*SEbabbb42(A)1000 ^MI Low CO2High CO21['Q Step-down(a3 Step-up*SE600800b400200aba•1480^120^140^202Age (days)Figure 6. Effects of CE on (a) root dry weight, and (b) totalseedling biomass of Engelmann spruce seedlings. The valuefor each bar is a mean of 24 seedlings. At each age, barsaccompanied by the same letter(s) or no letters were notsignificantly different at the P<0.01 level. For details seeFig. 5.5432143CI 0 ±SEEN Low CO 2High CO2Step-down22:1 Step-up -NN180^120^140^202Age (days)Figure 7. Effects of CE on shoot:root ratio (DW basis) ofEngelmann spruce seedlings. The value for each bar is a meanof 24 seedlings. At each age, bars accompanied by the sameletter(s) or no letters were not significantly different atthe P<0.01 level. For details see Fig. 5.444.1.1.2 Root Collar Diameter and Stem HeightRoot collar diameter of Engelmann spruce seedlingsresponded positively to CE at all stages of seedlingdevelopment except at 120 days (Fig. 8a). As in the case ofbiomass, the response to CE was more significant (P<0.001)after bud set (140 and 202 days), with high, step-down, andstep-up CO2 treatments being generally superior to low CO2treatment. Note the steady increase in root collar diameterafter bud set, particularly in seedlings grown under highCO2 regime. This steady increase in root collar diameterafter bud set and the cessation of shoot height, indicatesthat a portion of the available assimilates from continuingphotosynthesis were used in radial growth. In other words,in terms of size, the main effect of CE after bud set isthat seedlings became "stockier".The effects of CE on stem height of Engelmann spruceseedlings are presented in Fig. 8b. Data reported here areonly for seedlings grown under high and low CO2concentrations (i.e. controls) Data for step-down and step-up treatments were omitted because the trends were similarto controls. Data for 202 days were not taken because shootheight growth had ceased long before this date. Carbondioxide enrichment significantly (P<0.001) affected stemheight only at 140 days. However, at all previous harvests,even before 80 days (data not presented), high CO 2 seedlingsalways had a greater mean height than low CO 2 seedlings.11111 Low CO 2High CO2tSEbII^I1412(A)3.0,—.E 2.5EL.w 2.0.4iE06 1.5L.0o 1.0C.)"60ix 0.50.080^120^140^202Age (days)(B)4580^120^140Age (Days)Figure 8. Effects of CE on (a) root collar diameter, and (b) stemheight of Engelmann spruce seedlings. The value for each baris a mean of 24 seedlings. At each age, bars accompanied bythe same letter(s) or no letters were not significantlydifferent at the P<0.01 level. For details see Fig. 5.Overall, it appears that CE had a greater effect on biomassproduction than on stem height and root collar diameter ofEngelmann spruce seedlings.4.1.2 Carbohydrate ReservesThere was a general tendency for carbohydrate levels todecrease initially at 120 days, presumably due to thereduction in photoperiod (Figs. 9-16). Following this, asshoot growth was curtailed, reserves began to accumulate.Carbon dioxide enrichment significantly (P<0.001) increasedthe whole plant total non-structural carbohydrates (TNC)only after bud set (120 and 140 days in Fig. 9). Both thehigh and step-up CO2 treatments showed this increase in TNC.However, by the time seedlings were ready for cold-storage,TNC levels were the same in all treatments. After two andfour months (at 268 and 327 days, respectively) of storage,there was almost a one-third reduction in reserves. Thereduction in TNC during the two storage durations wascomparable, although the depletion was slightly more afterfour months storage.In terms of reserve allocation, roots contained about50% of the whole plant TNC just before storage (202 days),whereas, at all other times, needles had the highest4618—h day I Cold—storage9—h day1\\\1High CO 2Step-downEEO Step-upb±SEami Low CO 2b80^120^140^202 268 327Whole Plant47500400c)0) 3000)E200F-1 00Age (days)Figure 9. Trends in whole plant total non-structuralcarbohydrates (TNC) of Engelmann spruce seedlings. The valuefor each bar is a mean of 2-6 (at 268 days), 6 (at 202 and327 days), and 12 (at 80, 120, and 140 days) seedlings. Ateach age, bars accompanied by the same letter(s), or noletters were not significantly different at the P<0.01level.18—h day I Cold—storage9—h day32726880^120^140^202eTh 750E4-0RI 50F—Reserve AllocationAge (days)Figure 10. Trends in allocation of reserves to individual plantparts, expressed as a percentage of the maximum total non-structural carbohydrates (TNC) observed just prior tostorage (i.e. at 202 days). At each age, the proportion is atotal of all treatments representing 16 (at 268 days), 24(at 202 and 327 days), and 48 (at 80, 120, and 140 days)seedlings.4849proportion (Fig. 10). However, by the end of the two storagedurations, there was a considerable reduction (more than50%) in root TNC, and the needles at these times,constituted more TNC than any other plant part. Stemsrecorded the lowest allocation of reserves during the wholestudy period. Like needles, stem TNC percentages remainedfairly consistent. Respiration was presumably not restrictedto the roots and, in addition to the depletion of TNC duringstorage as an energy source for root respiration andmaintenance, it is logical to suspect that some root TNCmight have been translocated out of the roots (after starchconversion to glucose) to the needles and stems.During the whole study period, CO2 treatmentssignificantly (P<0.001) affected soluble sugar content inneedles at 140 days (Fig. 11), in stems at 80 days (Fig.12), and in roots at 80, 120 and 202 days (Fig. 13).Throughout the whole study period, particularly after budset, there was a general trend for high and step-up CO2treatments to contain more soluble sugars than the others.Overall, needles had the highest accumulation ofsoluble sugars, while stems and roots accumulated lower butcomparable amounts. In this study, it appears that all thethree plant parts of Engelmann spruce seedlings accumulatedand stored appreciable amounts of soluble sugars. For theneedles, soluble sugar content ranged from approximately 200to 350 mg g -1 DW, with 140 and 202 days recording theNeedles18—h day I^9—h day^ Cold—storage505000 400cn300rncn7 200ur)wD-6 100ylum Low CO 2High CO 2Step-downEKg Step-up±SERNb b 80^120^140^202^268^327Age (days)Figure 11. Trends in soluble sugar content of needles (budsincluded) of Engelmann spruce seedlings. The value for eachdays), 6 (at 202 and 327 days),days) seedlings. At each age,letter(s), or no letters werenot significantly different at the P<0.01 level.bar is a mean of 2-6 (at 268and 12^(at^80, 120, and 140bars accompanied by the same18—h day I Cold—storage.9—h day268^32780^120^140^202Low CO 2^ High CO 2^ Step-downEZ Step-up±SEStems515000 400cncn300cn= 200ul0.15-6 1000Age (days)Figure 12. Trends in the soluble sugar content of stems ofEngelmann spruce seedlings. The value for each bar is a meanof 2-6 (at 268 days), 6 (at 202 and 327 days), and 12 (at 80days, 120 days, 140 days) seedlings. At each age, barsaccompanied by the same letter(s), or no letters were notsignificantly different at the P<0.01 level.Roots18—h day^9—h day Cold — storage 52500C) 400Cr)3000rn200Ul075 1 00Low CO 2High CO 2Step-downEDE] Step-up±SEN\1b80^120^140^202^268^327Age (days)Figure 13. Trends in soluble sugar content of roots of Engelmannspruce seedlings. The value for each bar is a mean of 2 (at268 days), 2-6 (at 202 and 327 days), and 12 (at 80, 120,and 140 days) seedlings. At each age, bars accompanied bythe same letter(s), or no letters were not significantlydifferent at the P<0.01 level.highest values (Fig. 11). Interestingly, there was no majordepletion of soluble sugars in the needles after two andfour months cold-storage durations.In the case of stems and roots, soluble sugar contentranged from about 90 to 250 mg g-1 DW and the proportions inthese plant parts were fairly close to each other at mosttimes (Figs. 12 and 13). Soluble sugar content in the stemswas not affected by the two cold-storage durations, whereasin the roots there was a reduction of about 20%, and thisreduction was almost the same at both durations.In the case of starch, CO2 treatments had a significant(P<0.001) effect on needles, stems, and roots only at 140days (Figs. 14-16). Here also, both the high and step-up CO2treatments showed this increase in starch. Contrary to thetrends observed in soluble sugars, starch accumulation andstorage did not seem to take place equally in all the threeplant parts studied. Instead, roots were the major storageorgan for starch, followed by stems (Figs. 15 and 16). Inboth roots and stems, starch accumulation rose sharply afterbud set, the period during which CE was probably moreeffective. From Figs. 12 and 13, it can be noted that,during the earlier stages (e.g. 80 and 120 days), starchaccumulation in stems and roots of Engelmann spruceseedlings was minimal.53As for soluble sugars, maximum starch accumulation in18—h do 9—h day Cold—storagein Low CO 2High CO 2Step-down1221 Step-up±SEN\1Needles54500400c)cn 300Cr)E0 200-6510080^120^140^202^268^327Age (days)Figure 14. Trends in starch content of needles (buds included) ofEngelmann spruce seedlings. The value for each bar is a meanof 2-6 (at 268 days) , 6 (at 202 and 327 days) , and 12 (80,120, and 140 days) seedlings. At each age, bars accompaniedby the same letter(s), or no letters were not significantlydifferent at the P<0.01 level.500400c)cn300ci)E0 200100Stems18—h day I^9—h day^i^Cold—storageIN Low CO 2High CO 2Step-down[Kg Step-up±SE80^120^140^202^268^327Age (days)55r\\]1Figure 15. Trends in starch content of stems of Engelmann spruceseedlings. The value for each bar is a mean of 2-6 (at 268days), 6 (at 202 and 327 days), and 12 (80, 120, and 140days) seedlings. At each age, bars accompanied by the sameletter(s), or no letters were not significantly different atthe P<0.01 level.Cold—storage9—h day18—h daYI^I\Ias Low CO 2High CO 2Step-downEZ Step-upb b±SEM IMI IMORoofs56500cy)400cn30 0E_c0 20010080^120^140^202^268^327Age (days)Figure 16. Trends in starch content of roots of Engelmann spruceseedlings. The value for each bar is a mean of 2-6 (at 268days), 6 (at 202 and 327 days), and 12 (at 80, 120, and 140days) seedlings. At each age, bars accompanied by the sameletter(s), or no letters were not significantly different atthe P<0.01 level.57roots (ca. 500 mg g-1 DW) and stems (ca. 200 mg g-1 DW) wasobserved just before storage (202 days). In needles, themaximum accumulation of starch was observed at 80 days, andwas less than 100 mg g-1 DW (Fig. 14). The results vividlyshow that, for Engelmann spruce seedlings, needles do notfunction as a major storage organ for starch. In all theindividual plant organs, there was more than 50% reductionin starch content after two and four months storage, withthe needles recording only a minimal amount.4.1.3 Bud break and Root Growth PotentialIn this study, bud break was assessed as mean days tofirst bud break. None of the CO2 treatments had anysignificant effect on days to first bud break at either 268or 327 days at the P<0.01 level. At both ages, there were nological trends observed for the treatment effects. However,at 268 days, where soil temperature was considered as afactor, bud break was significantly (P<0.001) affected, withthe 3 °C soil temperature treatment recording a greaternumber of days to first bud break (ca. 15 days) than the 7and 11 °C soil temperature treatments (Table 1). On average,the lowest number of days to first bud break (ca. 10-12days) was observed at 327 days (Table 1). At 327 days, daysto first bud break were generally fewer than at 268 dayseven when seedlings were grown at the same soil temperature(e.g. 11 °C). The fewer days to first bud break observed at327 days could be explained by the fact that, with longer58Table 1. The effects of pre-storage CE on days to first budbreak of Engelmann spruce seedlings. Values are treatmentmeans of 6 seedlings (at 268 days) and 18 seedlings (at 327days). Values in brackets are standard errors. Stepdownl/step upl and step down2/step up2 represent seedlingstransfered at 60 and 120 days, respectively. Treatmentmeans at each soil temperature were not significantlydifferent at the P<0.01 level.At 268 daysSoil Low High Step Step upl Step Step up2Temp. downl down23 ° C 14.8 13.33 14.29 14.33 15.14 15.0(0.49) (0.67) (0.29) (0.33) (0.59) (0.68)7° C 13.67 13.71 12.67 12.56 14.25 14.33(0.33) (0.72) (0.42) (0.44) (0.25) (0.33)11 ° C 13.77 13.5 13.33 13.0 13.67 13.35(0.36) (0.72) (0.42) (0.45) (0.33) (0.44)At 327 days11 ° C 9.67 11.57 11.89 10.89 12.56 11.35(0.74) (0.95) (0.42) (0.57) (0.79) (0.57)59Table 2. The effects of pre-storage CE on post-storage RGP(number of new roots >5 mm long) of Engelmann spruceseedlings. Values are treatment means of 6 seedlings (at 268days) and 18 seedlings (at 327 days). Values in bracketsare standard errors. Step downl/step upl and stepdown2/step up2 represent seedlings transfered at 60 and 120days, respectively. Treatment means at each soiltemperature were not significantly different at the P<0.01level.At 268 daysSoil Low High Step Step upl Step Step up2Temp. downl down27 ° C 14.17 15.50 20.17 18.50 22.67 24.16(6.17) (5.82) (12.39) (9.18) (11.64) (11.64)11 ° C 20,16 64.5 53.00 47.83 51.33 68.67(6.99) (22.36) (24.77) (10.37) (9.28) (10.89)At 327 days11 °C 42.11 27.78 47.22 66.11 58.26 60.39(8.39) (5.97) (11.48) (8.38) (10.08) (10.00)cold-storage durations, seedlings had a greater opportunityto attain the necessary chilling, and thereafter, accumulateadequate heat units to permit the resumption of shootgrowth.Similarly, at both 268 and 327 days, RGP was notsignificantly affected by any of the CO2 treatments at theP<0.01 level (Table 2). However, at both ages, the low andhigh CO2 treatments consistently showed low RGP valuesrelative to step-down and step-up CO2 treatments. As for budbreak, at 268 days, where soil temperature was considered asa factor, root growth was significantly (P<0.001) affected,with the 7 °C soil temperature treatment recording extremelylow numbers of new roots (ca. 10-20) as compared to the 11 °Csoil temperature treatment (ca. 50-70) (Table 2). Unlike budbreak, RGP values for 268 and 327 days at 11 °C soiltemperature were comparable.4.2 Stable Carbon Isotopes as TracersWhole seedling isotope composition and carbohydratereserves before and after cold-storage are reported in Table3. The TNC in unlabelled and labelled seedlings were nearlythe same, implying that the procedures used for isotopelabelling did not differentially affect the carbohydratestatus of the seedlings. As with the CE experiment,carbohydrates were depleted by approximately 50% after fourmonths cold-storage.6061Table 3. Isotopic composition (expressed as 8 13C value) of wholeseedling total tissue and total nonstructuralcarbohydrates (TNC) before and after cold-storage.813C valuesStarch^Sugars(^)Total tissueTNC (mg/g DW)±SEPre-cold storageUnlabelled -21.89 -23.77 -25.25 394 ±39Labelled -34.09 -40.38 -33.43 439 ±23Post-cold storageUnlabelled -21.44 -24.99 -25.57 199 ±27Labelled -35.22 -38.84 -33.01 237 ±2262Table 4. Trends in isotopic composition (expressed as 8 13Cvalue) of new white roots, and calculated per centcontribution of reserve carbon to new root construction of"labelled" seedlings.Days after planting^Mean 813C (to) ±SE^% reserve carbonUnlabelled^Labelled^±SE (N)9 d * -36.78 100 (1)18 d -22.94 ±0.64 -30.26 ±1.02 52.9 ±6.9 (9)36 d -21.91 ±0.41 -24.47 ±0.76 18.3 ±5.0 (6)* Control seedlings at this date did not have any new roots.As desired, there were clear differences betweenunlabelled and labelled seedlings in starch, sugar, andtotal tissue 513 C values. There were also differences in813C of total tissue, but these were not as marked becauseunlike the bulk of the TNC, total tissue would contain largeamounts of carbon fixed prior to the labelling period.Futhermore, for each treatment, the 513C values werevirtually the same before and after cold-storage, indicatingthat cold-storage did not alter isotopic composition of theseedlings.Table 4 shows trends in the isotopic composition of newwhite roots, and calculated contributions of reserve carbonto new root construction. Results from this study indicatethat after 9 days, 18 days, and 36 days, planted Engelmannspruce seedlings contained respectively, 100, 53, and 18%old carbon in the new roots. However, these contributionsrepresent only a very small proportion of the availablereserves remaining after storage. This is because biomass ofnew roots (ca. 3.8 mg/seedling) was small relative to thesize of the reserve carbohydrate pool (ca. 237 mg/seedlingafter storage). Assuming new roots are composed principallyof carbohydrates (including cellulose), and are thereforeabout 40% carbon by dry weight, and knowing that after 36days, 18% of this carbon originated from reserves, it can becalculated that less than 1% of the reserve pool ended up inthese roots (only ca. 0.3% in fact). It is however, not63known where or how the rest of the reserve carbon was used.It is possible that some of the old carbon remained unused.Otherwise, much of it could have been, (1) used in rootrespiration during the construction of new roots, (2)translocated to other growing tissues (e.g. cambium, shootmeristems) and used in the resumption of shoot growth, or(3) used in various metabolic processes for repair and/ormaintenance of extant tissues.64655.0 DISCUSSION5.1 Biomass Accretion and PartitioningThis study has revealed that, elevated CO2 levelsinfluenced biomass accretion in individual tissues ofEngelmann spruce seedlings, and that these effects were moredistinct after bud set (Figs. 5 and 6a). In all treatments,the greater allocation of biomass to needles and/or leavesfollowed by roots in Engelmann spruce seedlings in thisstudy is in agreement with seedling biomass accretion forother species (Brown and Higginbotham 1986, Campagna andMargolis 1989, Hagem 1948, Sionit et al. 1985, and Hollinger1987).Similarly, the trends in total seedling biomass wereidentical to those for the individual plant parts, althoughhere the CO2 treatments were effective only after bud set(Fig. 6b). The increase in total seedling biomass has alsobeen observed in other commercial tree species. Forinstance, Sionit et al. (1985) reported that total dryweight increased by 56% in loblolly pine and 43% in sweetgumat 500 gL•L -1 CO2 compared with 350 gL•L -1 CO2. Working withblack spruce, Campagna and Margolis (1989) indicated thattotal seedling biomass was 30 and 14% greater at 925 and1100 11L•L -1 CO2, respectively, than control seedlings thatdid not receive CO2 enrichment. However, these authors foundno stimulation of growth after bud set. Such differences arenot surprising because it is known that the degree ofresponse to CE can differ with respect to species, duration66of exposure, and stage of development (Brown andHigginbotham 1986, Kramer 1981, Sionit and Kramer 1986,Sionit et al. 1985, Tolley and Strain 1984). Looking at Fig.6b, it can be noted that high CO2 treatment yielded thehighest total seedling biomass at all stages of development,even where there were no statistical differences amongtreatments, at any given date. This suggests that, overall,CE enhanced dry matter production of Engelmann spruceseedlings, and that the effects were simply more apparentafter bud set.The shoot:root ratio of Engelmann spruce seedlings inthe present study (Fig. 8) compares favourably with thatreported for other species (Campagna and Margolis 1989,Tolley and Strain 1984). In most CE studies with treespecies, shoot:root ratio usually remains unchanged orslowly decreases as development progresses (Sionit et al.1985). The present observations, however, show a sharpdecline in shoot:root ratio at the end of the growing season(202 days in Fig. 7), although the treatment means were notstatistically different. This decline in shoot:root ratiomay partly be explained by the fact that shoot height growthceases after bud set, whereas, root growth continues untillimited by low temperature. Also the fact that roots wererapidly filling up with reserve carbohydrates could accountfor much of the increase in root dry weight.675.2 Root Collar Diameter and Stem HeightIncreases in stem diameter in response to CE havepreviously been demonstrated in sweetgum and loblolly pine(Sionit et al. 1985), ponderosa pine seedlings (Surano eta/. 1986), and in sweetgum and loblolly pine seedlingsunder CO2-enriched air with high irradiance (Tolley andStrain 1984). However, as already pointed out, suchresponses to CE are species specific, with some speciesresponding more positively than others. The results of thisstudy (Fig. 8a) also depicted similar trends in increasedroot collar diameter in response to CE.In this study, CE only significantly affected stemheight of Engelmann spruce seedlings at 140 days (Fig. 8b).However, the general trend was that the high CO2 treatmentattained greater height growth than the low CO2 treatment atall stages of development. The present observations are inagreement with the findings of Campagna and Margolis (1989)using black spruce, Surano et al. (1986) using ponderosapine, and Sionit et al. (1984) using sweetgum and loblollypine seedlings. It appears therefore, that for most speciesstudied (the present study included), the effects of CE ongrowth are greater on biomass production than on stemdiameter and stem height.685.3 Carbohydrate ReservesThere is a broad body of literature indicating thatcarbohydrates (mostly starch and sugars) are the major formof food reserves for most woody plants (Duryea and McClain1984, Glerum 1980a, 1980b, Kramer and Kozlowski 1979,Loescher et al. 1990, Ritchie 1984, 1987). In conifers,lipids and proteins are also important reserve materials(Glerum 1980a, Kramer and Kozlowski 1979), but there hasbeen little work to establish just how important they are inrelation to carbohydrates (Glerum 1980b). In temperateclimates, reserve carbohydrates are a source of substratesfor respiration during storage, and for early respiration,growth and development occuring in the subsequent year(Loescher et al. 1990, Marshall 1985, Ronco 1973). Inconifers, reserve accumulation usually occurs in late summerand autumn (Gholz and Cropper, Jr. 1991, Glerum 1980b,Loescher et a/. 1990), and all plant parts function as sitesof reserve accumulation and storage, but not with equalimportance simultaneously (Glerum 1980b, Kramer andKozlowski 1979).Generally speaking,^the trends^in^levels^ofcarbohydrate reserves observed in this study for Engelmannspruce seedlings are relatively similar to the ones reportedfor other species. In the present study, there was a generaltendency for carbohydrate levels to decrease initially at120 days, presumably due to the reduction in photoperiod(Figs. 9-16). The shortened photoperiod might have causedthe reduction in reserve carbohydrates because whenphotoperiod was reduced, there would have been fewer hoursand hence less total light available for dailyphotosynthesis.In the case of TNC, CE was only effective after budset, and by the end of the growing season (202 days in Fig.9), the treatment effects were no longer apparent. The highTNC content seen at 202 days was mainly attributable tocorrespondingly large increases in starch in the roots andsoluble sugars in the needles (which will be elaboratedlater). Carbon dioxide enrichment appeared to promote therate of TNC accumulation following bud set, and this effectwas apparent in both the high and step-up CO2 treatments.This effect was also seen in both major reserve pools (i.e.free sugars in the needles and starch in the roots). If highand step-up CO2 treatments can promote TNC accumulationafter bud set, then it may be logical to provide CE afterbud set instead of limiting its application to only theseedling emergence stage. Furthermore, if reservecarbohydrates in any way promote early growth andestablishment of stored seedlings, and if these reserves aredepleted during storage, then it is sensible to assume thatany way in which TNC can be increased prior to storage mightimprove regeneration success. As indicated by this study,late season pre-storage CE might under some circumstancesbring about some improvement in the whole plant TNC. Thefindings of this study differ from those reported by6970Campagna and Margolis (1989), in that CE did notstatistically alter TNC in black spruce seedlings. Again,these differences might simply reflect species specificresponse (Sionit et al. 1985, Tolley and Strain 1984, Sionitand Kramer 1986). Another possible explanation for the lackof an effect on TNC in black spruce could be that theduration of the CE treatment after bud set was insufficient.In terms of reserve allocation within the plant, rootsconstituted about 50% of the whole plant TNC just beforestorage (202 days), whereas, at all other times, needles hadthe higher proportion (Fig. 10). This suggests that, by thetime seedlings were going into cold-storage, roots were themain storage organ. These results support the generalobservations and views by Abod and Webster (1991), Gholz andCropper, Jr. (1991), Glerum (1980b), Loescher et a/. (1990),and McCracken (1979) that the root system is the mainstorage organ for total carbohydrate reserves in most treespecies. The results of this study also conform to thefindings of Glerum (1980b) and of Loescher et al. (1990)that the relative importance of different plant parts forstorage of reserves varies at different stages ofdevelopment.71Similarly, significant differences were observed insoluble sugar and starch content in response to CE byneedles, stems and roots; particularly after bud set (Figs.11-16). Campagna and Margolis (1989) observed similareffects in black spruce seedlings, but only prior to budset. For soluble sugar content, needles recorded the highestvalues at all stages of development (Figs. 11-13). Solublesugar contents in the stems and roots were comparable,although roots had slightly superior values. Here also, thefindings of this study follow the trends in soluble sugarcontent reported elsewhere for other species (Abod andWebster 1991, Gholz and Cropper, Jr. 1991, Glerum 1980b,Kramer and Kozlowski 1979, Krueger and Trappe 1967).Starch content in all the three plant parts studied wasaffected by CE only at 140 days (Figs. 14-16), but starchaccumulation and storage did not take place equally indifferent plant parts at different stages of development.Instead, roots were the major storage organ for starch,followed by stems, and in both cases starch accumulationrose sharply after bud set, reaching the maximum valueachieved prior to cold-storage (202 days in Figs. 15 and16). The very high build-up of starch in the roots and stemsafter bud set may reflect relative changes in the productionand use of carbohydrates. Production would be expected toexceed use during this period since there is minimal growth,both above and below ground, while photosynthesis remainspositive until the end of the season (unless set back by72repeated frost). Hagem (1941), and Krueger and Trappe (1967)speculated that, as diameter growth stops and root activityslows down in late October, starch and sugar reserves beginto increase gradually because, as autumn progresses, cooltemperatures might reduce respiration. Furthermore, Alvik(1941) working with spruce and pine seedlings demonstratedthat the average daylight and prevailing winter temperaturesare usually sufficient to give a positive balance ofassimilation over respiration for the greater part of thewinter. Results presented here, where temperature was notreduced until 180 days, and yet carbohydrate reservesincreased steadily, indicate that low temperatures may notbe necessary to achieve positive carbon balance.The trends observed in Engelmann spruce seedlingsfollow closely those reported by Abod and Webster (1991),Glerum (1980b), Krueger and Trappe (1967), Loescher et al.(1990), and Reid et al. (1988). Based on the observations ofthis study, it appears that starch was the principal form ofcarbohydrate in the roots, whilst soluble sugars were moreprevalent in the shoot, particularly in the needles.5.4 Effects of Cold-storage on Carbohydrate ReservesLoss of carbohydrate reserves during cold-storage hasbeen widely studied in many temperate conifer species(Duryea and McClain 1984, Glerum 1980b, McCracken 1979,Ritchie 1982, 1984, 1987, Ronco 1972, 1973). These losseshave been implicated as being responsible for poor growth73and establishment of outplanted seedlings. Therefore, onejustification for storing seedlings at low temperaturebetween lifting and planting is to minimize respiratory lossof carbohydrate reserves (Cleary and Tinus 1980 cited byAbod and Webster 1991). According to Ritchie (1987), cold-storage affects photosynthesis and respiration in two ways.First, the absence of light stops photosynthesis and second,low temperature decreases the rate of respiration. The neteffect is that seedlings consume their supply of reservecarbohydrates in storage, but they do so very slowly. Asstated by Duryea and McCracken (1984), Glerum (1980a), andKozlowski and Kramer (1979), the primary use of carbohydratereserves is to maintain respiration and growth when currentphotosynthate is not available.In this study, soluble sugars, starch and TNC were allaffected by the two storage durations, but to varyingdegrees. On average, all the CO2 treatments behaved the sameduring the two storage durations. No effects were expectedgiven the fact that all the CO2 treatments had almost thesame TNC level when going into storage. For TNC, there wasalmost a one third reduction after two and four months (at268 and 327 days in Fig. 9, respectively) in storage.Similar trends in TNC depletion have been observed inEngelmann spruce (Ronco 1972, 1973), in Mugo pine andradiata pine (McCracken 1979), in Douglas-fir (Ritchie 1982,1987), and loblolly pine (Reid et a/. 1988) seedlings.However, it is interesting to note that, in this study,74there was a much greater apparent depletion of carbohydratereserves between zero and two months storage than betweentwo and four months storage. One possible reason for such atendency in this study is that, when the cold-roomtemperature rose to about 10 °C for two days at 230 days,seedlings might have respired excessively, thereby utilizinga larger portion of their reserves. However, it is likelythat more reserve depletion occurred during the one weekthawing period (at 5 °C) just before planting. There was nosuch thawing period at 0 months storage as there was no needfor one. In a study with spruce and pine seedlings, Alvik(1941) reported that respiration increased sharply between0 ° and 10 °C, and attained a temperature quotient (Q10) of 2-3. A change in Q 10 much greater than this would be requiredto account for the depletion observed in the present study.Still a temperature rise from -5 °C (during storage) to 5 °C(during thawing) might have increased the respiration rateof the seedlings substantially. The suspicion therefore,that thawing may contribute to the depletion of reservecarbohydrates, may suggest the need to examine the lengthand temperature of the thawing period for effects on thecarbohydrate status of cold-stored seedlings. Changes in theQ10 for respiration at low temperature should be preciselyestablished.Comparing plant parts, by the end of the two storagedurations, roots had the highest reduction (more than 50%)in TNC and at these times, needles constituted more TNC than75any other plant tissue (Fig. 10). This is in agreement withother investigators who found similar reduction in root TNCafter cold-storage (Philipson 1988, McCracken 1979, Ritchie1982). On the whole, it appears that stem TNC was notaffected by the two storage durations, and that stem TNCremained fairly constant throughout the whole study period(Fig. 10).Starch content within plant tissues was significantlyaffected by the two and four months storage durations, andthe depletion of starch during the two storage durations wascomparable (at 268 and 327 days in Figs. 14-16). In all thethree plant parts studied, there was more than 50% starchdepletion as compared with the starch levels prior to cold-storage (i.e. at 202 days). While the results of this studyconform to those of Ronco (1972, 1973) for Engelmann spruce,and McCracken (1979) for Mugo and radiata pines, conflictingresults were reported by Krueger and Trappe (1967), andRitchie (1982) for Douglas-fir seedlings. Both of thesestudies indicated an increase in starch content betweenMarch and April (Krueger and Trappe 1967), and after ninemonths storage (Ritchie 1982).As regards soluble sugars, this study has shown thatthe two and four months storage durations did not have muchimpact on their depletion (at 268 and 327 days in Figs. 11-13). There was only about 5 and 25% reduction in needle androot soluble sugars, respectively. In studies with Engelmannspruce (Ronco 1972, 1973), Douglas-fir (Ritchie 1982,76Krueger and Trappe 1967), and Mugo and radiata pines(McCracken 1979), the pattern in the depletion of solublesugar content in needles, stems, and roots was essentiallyidentical to the results of the present study. The onlydifference was that, in this study, soluble sugar contentseems to have been maintained at the expense of starch,whereas, in the former experiments soluble sugar contentswere not static and declined almost linearly with storageduration. Furthermore, since respiration presumably occurredthroughout the plant and seedlings were notphotosynthesizing while in storage, it is reasonable toassume that the maintenance of needle sugar content was dueto starch conversion in roots to soluble sugars, which werethen translocated to the needles. This observation suggeststhat, at least in Engelmann spruce, efficient phloemtransport between the roots and needles may occur instorage. The enzymatic reaction involved in maintaining theequilibrium between starch and free sugars is somewhattemperature dependent. Low temperature favours conversion ofstarch to free sugars (ap Rees et al. 1988, DeLucia 1986,Ritchie 1982), but the actual control of this process is notwell understood. Specific experiments should be designed toexamine circumstances surrounding starch conversion andphloem transport in Engelmann spruce.On the whole, the results from this study stronglyconform to the already established fact that carbohydratelevels of cold-stored seedlings deplete with storage77duration. However, there are some differences in the levelof depletion depending on the individual species, liftingdate, storage temperature, storage duration, and othercultural practices (Duryea and McClain 1984). This studyalso indicates that pre-storage CE per se did not have anyinfluence on the degree of TNC, starch and soluble sugardepletion in Engelmann spruce seedlings while in storage.5.5 Bud BreakThe data reported in this study indicate that none ofthe CO2 treatments showed any significant influence on thedays to first bud break at either 268 or 327 days (Table 1).However, at 268 days, where soil temperature was consideredas a factor, bud break was significantly affected by soiltemperature. It is important to point out here that,although soil temperatures differed statistically, the meandays to first bud break did not differ greatly, especiallybetween 7 and 11 °C soil temperatures. These results supportthe findings of Cam and Harper (1991) who reported similartrends in days to terminal bud break in white spruceseedlings after 15 weeks of cold-storage.Results from soil temperature studies on Douglas-fir,Pacific silver fir (Abies amabilis (Dougl.) Forbes), noblefir (Abies procera Rehd), lodgepole pine, and ponderosa pineseedlings (Lopushinsky and Max 1990) also showed decreasingdays to bud break with increasing soil temperature. However,even with as wide as 0-30 °C soil temperature range the true78firs did not respond as strongly to the increasing soiltemperature as did Douglas-fir and the pines. Earlier workby Lavender and Overton (1972) reported that the reductionin shoot growth of Douglas-fir seedlings associated with lowsoil temperature was not occasioned by reduced water ormineral uptake. Their alternative explanation was that whenroots are grown in cold soils, shoot growth or bud activityis slowed by reduced export, from the roots, of some plantgrowth regulatory substance or substances (presumablygibberellins). Similar conclusions were reached by Lavenderand Wareing (1972), and Lavender et al. (1973) who foundthat in Douglas-fir transplants, gibberellin-like compoundswere synthesized in the roots and then exported to theshoots where they enhanced bud activity.The decrease in days to first bud break with increasingcold-storage duration is also in conformity with what otherresearchers have reported. For example, Camm and Harper(1991) working with white spruce found a similar pattern indays to bud break over a wide range of cold-storagedurations (0-30 weeks). Similarly, several authors (Burr etal. 1989, Carlson 1985, Ritchie 1984, Ritchie et al. 1985,van den Driessche 1977) working with a wide range of speciesalso reported decreasing days to terminal bud break withincreasing storage duration. This decrease in days to budbreak may reflect the accumulation of chilling hoursrequired to fully release dormancy. Chilling requirements inspruce are usually satisfied after 6-8 weeks of exposure to79low temperatures (ca. 4-6 °C) (Lavender unpublshed data,Nienstaedt 1966, 1967). As noted by many researchers (Burret al. 1989, Carlson 1985, Lavender 1985, Ritchie and Dunlop1980, Ritchie et al. 1985), accumulation of chilling hoursrequired to release dormancy for most temperate coniferspecies can be achieved by placing seedlings in storageafter they have entered rest, usually in late November orearly December. It is also known that chilling interruptionby brief periods of warmer temperature during storage canaffect dormancy release, and that the degree of negating thechilling response depends on timing, duration, andtemperature during the interruption period (Lavender:personal communication, van den Driessche: cited by Ritchie1984,). In this study, a brief interruption of cold-storageat 230 days (i.e. 10 °C for two days) may have influenceddays to first bud break.Early bud break may be beneficial at locations withshort growing periods (e.g. interior B.C.), in that,outplanted seedlings can resume growth early to takeadvantage of the short favourable growing season. Equallyimportant, a delay in dormancy release during cold-storagecan be used to the advantage of a practising forester insignificantly expanding the planting "window". However, onemust be cautious to ensure that any storage duration orprotocol chosen does not significantly promote depletion ofcarbohydrate reserves. As already indicated in this study,and by numerous researchers, prolonged storage durationsnegatively affect carbohydrate levels in most forest treeseedlings. It has not, however, been well-established, hereor elsewhere, as to what degree of reserve depletion istolerable. Substantial, almost complete loss of reserves maybe inconsequential (Marshall 1985, Omi and Rose 1990,Ritchie 1982, 1984, Ronco 1973), but complete exhaustion ofreserves is very likely fatal.5.6 Root Growth PotentialAlthough it has long been suspected that carbohydratereserves are important for outplanted seedlings afterstorage, the direct relationships between reserves and earlyroot growth after cold-storage have not been thoroughlyexplored. There are still numerous conflicting results asto whether new root growth after cold-storage depends on"old" carbon (stored carbohydrates) or "new" carbon (currentphotosynthate) or both.5.6.1 Effects of CE and Soil Temperature on RGPIn the present study, none of the CO2 treatments hadany positive effects on RGP of Engelmann spruce seedlings atboth 268 and 327 days (Table 2). Since all the CO2treatments had almost the same TNC level prior to cold-storage, it was not surprising, at least in terms of reserveutilization, that RGP was not affected by CE after storage.On the other hand, the significant effect observed at 268days in response to varying soil temperatures is in8081agreement with the results reported by many investigators.In a study with white spruce, Camm and Harper (1991)indicated that, after 17.5 weeks of cold-storage, initiationof new root growth was soil temperature-dependent. Theresults reported in this study compare favourably with thoseof Camm and Harper (1991) for white spruce, and are abovethe RGP threshold value for adequate root regeneration (10new roots > 10mm long) for interior spruce and lodgepolepine reported by Simpson et al. (1988). Reduced root growthat low soil temperatures is not surprising because similarfindings have been reported for other conifers. In anexperiment with Douglas-fir, Pacific silver fir, noble fir,lodgepole pine, and ponderosa pine transplants, Lopushinskyand Max (1990) indicated that maximum root growth in allspecies occurred at 20 °C. Furthermore, soil temperatures of10 °C or less drastically reduced or prevented new rootgrowth. Other studies with Douglas-fir seedlings(Lopushinsky and Kaufmann 1984, Ritchie 1985) also showedthat little root growth occurred at soil temperatures of 5 °Cor less. In contrast, Lavender and Wareing (1972) reportedimproved root growth in Douglas-fir seedlings at 4 °C soiltemperature as a result of application of gibberellin-likecompounds. If rapid initiation of new root growth isessential for the early establishment of outplantedseedlings, and if new root growth does not generally proceedwell at soil temperatures below 10 °C, it is logical tosuggest that seedlings should not be planted until soils82warm up to 10 °C or more. However, in many situations,waiting for the soil temperature to warm up to 10 °C. wouldmean extending storage duration which in turn may affect theseedling quality for a variety of reasons, including reservedepletion. Another possibility would be to move towardssummer lifting/and planting (i.e. so-called "hot" planting).The development of RGP in conifer species tends tofollow a general pattern; low in fall, reaching a peak inmid-winter, declining in spring, and then rising slightlyagain in late summer (Ritchie 1985, Ritchie and Dunlop 1980,Ritchie and Tanaka 1990, Stone and Jenkinson 1970). Inaddition to soil temperature and physiological condition(particularly dormancy status), cold-storage in interactionwith lifting date may also have a strong effect on theexpression of RGP (Ritchie 1985). Generally speaking,seedlings lifted in mid-winter tend to have higher RGP thanfall or spring-lifted seedlings (Burdett 1987, Ritchie andDunlop 1980, Ritchie et al. 1985, Silim and Lavender 1992,Sutton 1990). They also have higher RGP following storagefor a few months.5.6.2 Effects of Cold-storage on RGPIn this study, there was no clear pattern in the RGP ofEngelmann spruce seedlings with different storage durationas assayed at a soil temperature of 11 °C (Table 2). Theresults from this study therefore are not consistent withthe general contention that, for most conifer species, RGP83increases during the initial stages of cold-storage, andthen declines with prolonged cold-storage duration. On thewhole, RGP values are notoriously variable, and trendsobserved between and even within different studies are ofteninconsistent, partly due to differing test environments,management practices, and methods of evaluating RGP (Ritchieand Dunlop 1980, Ritchie et a/. 1990, Sutton 1990). Forinstance, Carlson (1985) working with loblolly pineseedlings found that RGP in fall-lifted seedlings wasreduced during cold-storage, whereas storage after mid-winter (after 734 chilling hours) either improved or did notaffect RGP. Ritchie et a/. (1985) studied RGP in lodgepolepine and interior spruce seedlings, and in both species,December to March lifting dates, followed by a two monthstorage had little effect on RGP. However, when the sameseedlings were subjected to a six month storage, RGP wassubstantially reduced especially for the March lift date. Inseparate experiments, Camm and Harper (1991) and Ritchie(1982), working with white spruce and Douglas-fir seedlingsrespectively, observed that RGP steadily increased duringthe initial stages of cold-storage (ca. 0-4 months), butdeclined sharply after prolonged storage.The physiological condition of the seedlings at theinitiation of storage could be viewed as a major factorcontrolling RGP periodicity. For example, Ritchie and Dunlop(1980) speculated that seasonal patterns in carbohydratesynthesis, storage, conversion, and metabolism may be theprimary modulators of RGP periodicity in trees. They furtherstated that the highest concentration of carbohydratesduring mid-winter happens to coincide with the presumed RGPand stress resistance peaks, after which RGP declines ascarbohydrate reserves deplete during storage. Thiscoincidence between carbohydrate levels and RGP may haveencouraged the concept that carbohydrate reserves areimportant substrates for root growth.5.5.3 Role of Carbohydrates in RGPSeveral studies on the role of carbohydrate reserves inRGP have presented conflicting results. However, thedependence of outplanted seedlings on either carbohydratereserves, current photosynthate or a combination appears tobe somewhat species specific. The results from this studyindicate that after 9, 18, and 36 days, the new roots ofplanted seedlings of Engelmann spruce seedlings wererespectively, comprised of 100, 53, and 18% "old" carbon(Table 4). This suggests that, at least during the first 9days, new root growth of planted Engelmann spruce seedlingsdepends entirely on carbohydrate reserves; although therewas very little root growth during this initial period.There is no doubt, however, that reserve carbon didcontribute significantly to root construction up to 36 daysafter planting. These observations support the contention ofmany authors that new root growth in some species depends,at least in part, on carbohydrate reserves. For instance, in8485disbudding and girdling or bark-ringing experiments,Philipson (1988) reported that new spring root growth ofSitka spruce transplants depended on carbohydrates storedwithin the root. By way of radioisotope experiments, van denDriessche (1987) obtained contradictory results; new rootgrowth for both Douglas-fir and Sitka spruce seedlingsseemed to depend on current photosynthate. Lavender andHermann (1970), and Lavender and Wareing (1972) alsoreported that new root growth of Douglas-fir transplants maylargely depend on some substance or substances, presumablyhormones or carbohydrates, exported from the foliage. Thesefindings match with those of Ursino et al. (1968), who alsoreported that the new roots of white pine (Pinus strobus L.)transplants received current photosynthate from the shootthroughout the entire growing season.However, neither girdling nor radioisotope experimentscan provide unequivocal results useful for finally resolvingthe controversy. For example, bark-ringing experiments maynot only exclude translocation of current photosynthate downto the root system, but other known or unknown compounds aswell. As noted by Philipson (1988), although new root growthin Sitka spruce seedlings was not dependent on currentphotosynthate, after 14 days, bark-ringed trees started toshow retarded growth, suggesting that the roots were lackingsome factor. The main shortfall in using radioisotopes forC-labelling is that the labelling period is short (rangingfrom one hour to a couple of days) and, as such, may notprovide a uniform or thorough labelling of reserves. Stablecarbon isotope labelling, as used in this study shouldprovide a better technique for labelling and then tracingthe allocation of "old" carbon to a given plant part (newroots in this case).In girdling experiments it is very difficult toimplicate lack of current photosynthate as the sole factorinhibiting new root growth. It has been speculated by manyresearchers that certain unknown compounds (presumably plantgrowth regulators), which are translocated along withcurrent photosynthate to the roots also play some role innew root growth (Deyoe and Zaerr 1976, Kramer and Kozlowski1979, Philipson 1988, Ritchie and Dunlop 1980, Zaerr andLavender 1974). Van den Driessche (1991) investigated theeffect of low CO2 concentration, darkness, and stem girdlingon new root growth in Douglas-fir seedlings. He found thatnew root production was affected by the treatments in theorder: control > low CO2 concentration > dark > girdling.Production of new roots was limited in the absence ofphotosynthesis (i.e. dark treatment) but was even moreseriously affected by girdling, suggesting that lack ofcurrent photosynthate and presumably other factors mighthave inhibited new root growth. His findings supported theviews of Lavender and Hermann (1970), Philipson (1988),Ritchie (1982), van den Driessche (1987), and Zaerr andLavender (1974) that darkening or girdling almost completelyprevents new root production. Girdling treatment was more86severe in inhibiting new root production presumably becauseonly reserves stored in the root system would be accessible,whereas, in the low CO2 concentration and dark treatments,reserves throughout the whole plant would have beenaccessible.876.0 CONCLUSIONSThe principal objective of this thesis research was toestablish the role of carbohydrate reserves in spring rootgrowth of freezer-stored Engelmann spruce seedlings.Emphasis was placed on investigating the utility andpossible side effects of CE in the manipulation of thesereserves. Based on the foregoing results and discussion, thefollowing salient conclusions can be drawn from the presentstudy:1. Biomass production by individual plant parts in Engelmannspruce seedlings was more significantly affected than rootcollar diameter and stem height by CE. These effects weremore apparent after bud set.2. Despite the fact that biomass production of individualplant parts was affected by CE, shoot:root ratio was notaffected by CE, and gradually decreased during the studyperiod (i.e. 80-202 days).3. Carbon dioxide enrichment had little significant effecton carbohydrate levels prior to bud set.4. In contrast to the above, CE did promote the rate ofcarbohydrate accumulation following bud set, but had noinfluence on the ultimate level attained just beforestorage.885. After two and four months storage, there was almost aone-third reduction in the whole plant TNC.6. Soluble sugars in all plant parts were not significantlyaffected by either two or four months storage whereas, therewas more than 50% reduction in starch in all plant partsfollowing two and four months storage.7. Carbon dioxide enrichment did not have any detectableeffect on days to bud break or RGP.8. Low soil temperature increased the number of days to budbreak and adversely affected RGP.9. Stored reserves made an important contribution to earlyspring root growth, but this sink was only a minor drain onthe reserve pool.897.0 RECOMMENDATIONSThe results from this study may be of some practicaluse in the reforestation program in B.C. (especiallynortherly areas) in the following ways:1. In most of the parameters measured (i.e. biomassproduction, growth variables, and carbohydrates), theeffects of CE were most apparent after bud set. Therefore,nursery operators should consider CE of Engelmann spruceseedlings applied after bud set to benefit growth andmanipulate seedling morphology. Carbon dioxide enrichmentfollowing bud set would effectively increase biomassindependently of shoot height growth.2. Extended storage and/or thawing periods should beavoided as much as possible. As noted by Marshall (1985),and Omi and Rose (1990) the degree of carbohydrate depletionduring storage will depend on the amount of reserves goinginto storage and subsequent storage duration andtemperature. There is some potential for the use of CO2enrichment for enhancement of reserve levels prior tostorage.903 Future ResearchOwing to time constraints and the fact that factorsaffecting root regeneration, especially in cold soils arediverse, it was not feasible to tackle all aspects relatedto root regeneration in this one thesis project. Therefore,the following are some areas in need of future research:- The effects of thawing period and temperature on thedepletion of carbohydrates following cold-storage, should beinvestigated.- The storage period should be extended to six months asinitially planned in the present study to cover a morecomplete range of storage durations that might demonstrateadverse effects of prolonged cold-storage in terms ofcarbohydrate depletion and expression of RGP.- The observed effects of CE after bud set should beinvestigated for possible impacts on seedling fieldperformance.- The role of roots as a major site of storage suggests thatmore emphasis should be placed on understanding roothardiness, mobilization of root reserves, etc.91- The CE experiment should be repeated. This should be doneafter bud set for a shorter duration (ca. 6-7 weeks). 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Biostatistical Analysis (Second edition).Prentice Hall, Englewood Cliffs, NJ. 718 p.102Appendix 1 ANOVA for the effect of CE on needle, stem, androot dry weights in Engelmann spruce seedlings.(A) Needle dry weight at 80 days.SV^SS^DF^MS^F -Ratio PTreatments^73154.533^3^24384.844^3.010^0.034Error^745388.366^92 8102.047(B) Stem dry weight at 80 days.SV^SS^DF^MS^F-Ratio PTreatments^8892.862^3^2964.287^4.683^0.004Error^58231.422^92 632.950(C) Root dry weight at 80 days.SV^SS^DF^MS^F-RatioTreatments^31948.815^3^10649.605^6.853^0.000Error^142964.422^92 1553.961(D) Needle dry weight at 120 days.SV^SS^DF^MS^F-RatioTreatments 244066.294^3^81355.431^1.958^0.126Error^3823036.971^92^41554.750(E) Stem dry weight at 120 days.SV^SS^DF^MS^F-RatioTreatments^180.495^3^14393.498^2.083^0.108Error^635763.184^92 6910.469103104(F) Root dry weight at 120 days.SV^SS^DF^MSTreatments^69206.005^3^23068.668Error^1257376.160^92^13667.132(G) Needle dry weight at 140 days.SV^SS^DF^MSF -Ratio1.688F -Ratio0.175PTreatments^680709.781 3^560236.594 11.328 0.000Error^4549861.254 92^49455.01(H) Stem dry weight at 140 days.SV^SS DF^MS F -RatioTreatments^387515.884 3^129171.961 9.819 0.000Error^1210320.649 92^13155.659(I) Root dry weight at 140 days.SV^SS DF^MS F -RatioTreatments^837655.114 3^279218.371 10.823 0.000Error^2373570.919 92^25799.684(J) Needle dry weight at 202 days.SV^SS DF^MS F -RatioTreatments^734801.893 3^244933.964 8.456 0.001Error^695197.634 24^28966.568(K) Stem dry weight at 202 days.SV^SS DF^MS F -Ratio PTreatments^167519.070 3^55839.690 4.903 0.008Error^273318.020 24^11388.251(L)^Root dry weight at 202 days.SV SS DF^MS F -RatioTreatments^449830.473 3^149943.491 2.687 0.069Error^1339381.737 24^55807.572105Appendix 2^ANOVA for the effect of CE on total biomassproduction in Engelmann spruce seedlings.(A) Total seedling biomass at 80 days.SV^SS^DF^MS^F-Ratio^PTreatments^277816.235^3^92605.412^4.620^0.005Error^1843997.912 92^20043.456(B) Total seedling biomass at 120 days.SV^SS^DF^MS^F-Ratio^PTreatments 810464.168^3^270154.723^1.980^0.122Error^.125533E+08^92^136449.202(C) Total seedling biomass at 140 days.SV^SS^DF^MS^F-Ratio^PTreatments 8062351.407^3 2687450.469^12.478^0.000Error^.198144E+08^92^215373.882(D) Total seedling biomass at 202 days.SV^SS^DF^MS^F-Ratio^PTreatments 3351344.275^3 1117114.758^8.258^0.001Error^3246539.314^24^135272.471106Appendix 3 ANOVA for the effect of CE on shoot:rootratios in Engelmann spruce seedlings.(A) Shoot:root ratios at 80 days.SV^SS^DF^MS^F-RatioTreatments 5.001^3^1.667^2.512^0.063Error^61.064^92 0.664(B) Shoot:root ratios at 120 days.SV^SS^DF^MS^F-RatioTreatments^9.270^3^3.090^6.478^0.001Error^43.887 92 0.477(C) Shoot:root ratios at 140 days.SV^SS^DF^MS^F-RatioTreatments^3.401^3^1.134^1.884^0.138Error^55.356 92 0.602(D) Shoot:root ratios at 202 days.SV^SS^DF^MS^F-RatioTreatments 1.538^3^0.513^1.610^0.209Error^8.917 28 0.318107Appendix 4 ANOVA for the effect of CE on diameter and stemheight growth in Engelmann spruce seedlings.(A) Diameter growth at 80 days.SV^SS^DF^MS^F-RatioTreatments 0.418^3^0.139^4.469^0.006Error^2.869^92^0.031(B) Diameter growth at 120 days.SV^SS^DF^MS^F-RatioTreatments 0.534^3^0.178^1.531^0.212Error^10.684^92^0.116(C) Diameter growth at 140 days.SV^SS^DF^MS^F-RatioTreatments 4.173^3^1.391^9.334^0.000Error^13.710^92^0.149(D) Diameter growth at 202 days.SV^SS^DF^MS^F-RatioTreatments 0.564^3^0.188^5.028^0.008Error^0.898^24^0.037(E) Stem height growth at 80 days.SV^SS^DF^MS^F-RatioTreatments 3.968^1^3.968^2.099^0.154Error^86.943^46^1.890(F) Stem height growth at 120 days.SV^SS^DF MS^F-RatioTreatments 12.403^1 12.403^1.538^0.221Error^371.069^46^8.067108(G) Stem height growth at 140 days.SV^SS^DF MS^F-Ratio^PTreatments 93.800^1^93.800^12.003^0.001Error^359.490^46^7.815Appendix 5 ANOVA for the effect of CE on carbohydratereserves at 80 days.(A) Starch in needles.SV^SS DF MS F-Ratio PTreatments^14735.095 3 4911.698 1.993 0.129Error^108447.059 44 2464.706(B) Starch in stems.SV^SS DF MS F-Ratio PTreatments^747.122 3 249.041 2.143 0.108Error^5114.314 44 116.234(C)^Starch in roots.SV SS DF MS F-Ratio PTreatments^941.146^3^313.715^1.174^0.331Error^11759.898^44^267.270(D) Soluble sugars in needles.SV^SS^DF^MS^F -Ratio^PTreatments^36137.536^3^12045.845^1.463^0.238Error^362206.300^44^8231.961(E) Soluble sugars in stems.SV^SS^DF^MS^F-Ratio^PTreatments^38420.142^3^12806.714^9.088^0.000Error^62002.888^44^1409.157(F) Soluble sugars in roots.SV^SS^DF^MS^F-RatioTreatments 104512.767^3^34837.589^24.651^0.000Error^62181.038^44^1413.205(G) Whole plant TNC.SV^SS^DF^MS^F-RatioTreatments^6214.097^3^2071.366^0.478^0.699Error^190637.767^44^4332.677Appendix 6 ANOVA for the effect of CE on carbohydratereserves at 120 days.(A) Starch in needles.SV^SS^DF^MS^F-Ratio^PTreatments^1670.615^3^556.872^2.242^0.097Error^10929.628^44^248.401(B) Starch in stems.SV^SS^DF^MS^F-RatioTreatments^257.559^3^85.853^1.865^0.149Error^2025.193^44^46.027(C) Starch in roots.SV^SS^DF^MS^F-RatioTreatments^211.456^3^70.485^0.626^0.602Error^4952.226^44^112.551(D) Soluble sugars in needles.SV^ SS^DF MS^F-RATIOTreatments^35034.436^3 11678.145^4.097^0.012Error^125431.529^44^2850.717109(E) Soluble sugars in stems.SV^SS^DF^MS^F-RATIO^PTreatments^20985.172^3^6995.057^3.842^0.016Error^80108.979^44^1820.659(F) Soluble sugars in roots.SV^SS^DF^MS^F-RATIOTreatments^8039.302^3^2679.767^5.509^0.003Error^21402.470^44^486.420(G) Whole plant TNC.SV^SS^DF^MS^F-RatioTreatments^21564.213^3^7188.071^5.571^0.002Error^56770.012^44^1290.228Appendix 7 ANOVA for the effect of CE on carbohydratereserves at 140 days.(A) Starch in needles.SV^SS^DF^MS^F-RatioTreatments^17699.206^3^5899.735^4.931^0.005Error^52647.756^44^1196.540(B) Starch in stems.SV^SS^DF^MS^F-RatioTreatments^13241.021^3^4413.674^6.916^0.001Error^28080.447^44 638.192(C) Starch in rootss.SV^SS^DF^MS^F-RatioTreatments^89677.812^3^29892.604^9.478^0.000Error^138766.489^44^3153.784110111(D) Soluble sugars in needles.SV^SS^DF^MS^F-Ratio^PTreatments 189912.464^3^63304.155^14.727^0.000Error^189135.203^44^4298.527(E) Soluble sugars in stems.SV^SS^DF^MS^F-RatioTreatments^19322.194^3^6440.731^2.707^0.057Error^104680.250^44^2379.097(F) Soluble sugars in roots.SV^SS^DF^MS^F-RatioTreatments^11484.508^3^3828.169^3.488^0.023Error^48292.811^44^1097.564(G) Whole plant TNC at 140 days.SV^SS^DF^MS^F-RatioTreatments^171572.186^3^57190.729^19.590^0.000Error^128451.948^44 2919.362Appendix 8 ANOVA for the effect of CE on carbohydratereserves at 202 days.(A) Starch in needles.SV^SS DF MS F-RatioTreatments^4264.294 3 1421.431 2.070 0.130Error^17165.508 25 686.620(B) Starch in stems.SV^SS DF MS F-RatioTreatments^961.970 3 320.657 0.196 0.898Error^40891.470 25 1635.659(C) Starch in roots.SV^SS^DF^MS^F-RatioTreatments^4793.192^3^1597.731^0.261^0.853Error^153042.129^25^6121.685(D) Soluble sugars in needles.SV^SS^DF^MS^F-RatioTreatments^27341.579^3^9113.860^3.280^0.037Error^69460.530^25^2778.421(E) Soluble sugars in stems.SV^SS^DF^MS^F-Ratio^PTreatments^2750.799^3^916.933^3.381^0.034Error^6779.269^25^271.171(F) Soluble sugars in roots.SV^SS^DF^MS^F-RatioTreatments^32930.876^3^10976.959^11.803^0.000Error^23250.298^25 930.012(G) Whole plant TNC.SV^SS^DF^MS^F-RatioTreatments 10358.780^3^3452.927^2.109^0.125Error^40935.990^25^1637.440Appendix 9 ANOVA for the effect of CE on carbohydratereserves at 268 days.(A) Starch in needles.SV^SS^DF^MS^F-Ratio^PTreatments^10.131^3^3.377^3.163^0.064Error^12.813^12^1.068112(B) Starch in stems.SS^DF^MS^F-Ratio^PTreatments 1877.587^3^625.862^0.818^0.508Error^9178.559^12^764.880(C) Starch in roots.SS^DF^MS^F-Ratio^PTreatments 8489.077^3^2829.692^0.771^0.532Error^44061.791^12^3671.816(D) Soluble sugars in needles.SV^SS^DF^MS^F-Ratio^PTreatments 6849.386^3^2283.129^2.131^0.150Error^12856.853^12^1071.404(E) Soluble sugars in stems.SV^SS^DF^MS^F-Ratio^PTreatments 2925.569^3^975.190^2.822^0.084Error^4147.044^12^345.587(F) Soluble sugars in roots.SV^SS^DF^MS^F-RATIO^PTreatments^13713.960^3^4571.320^3.547^0.048Error^15467.311 12^1288.943(G) Whole plant TNC.SS^DF^MS^F -Ratio^PTreatments^9915.705^3^3305.235^1.957^0.174Error^20264.445^12^1688.704SVSVSV113114Appendix 10 ANOVA for the effect of CE on carbohydratereserves at 327 days.(A) Starch in needles.SV^SS^DF^MS^F-RatioTreatments^8.870^3^2.957^4.024^0.022Error^14.696^20^0.735(B) Starch in stems.SV^SS^DF^MS^F-RatioTreatments^760.760^3^253.587^0.175^0.912Error^8910.440^20^1445.522(C) Starch in roots.SV^SS^DF^MS^F-Ratio^PTreatments^3671.468^3^1223.823^0.670^0.580Error^36507.081^20^1825.354(D) Soluble sugars in needles.SV^SS^DF^MS^F-RatioTreatments^5410.845^3^1803.615^4.960^0.010Error^7272.851^20 363.643(E) Soluble sugars in stems.SV^SS^DF^MS^F-RatioTreatments^5710.163^3^1903.388^1.625^0.215Error^23423.801^20^1171.190(F) Soluble sugars in roots.SV^SS^DF^MS^F-RatioTreatments^5421.281^3^1807.094^1.231^0.325Error^29362.064^20^1468.103115(G) Whole plant TNC.SV^ SS^DF^MS^F-RatioTreatments^3711.179^3^1237.060^1.580^0.225Error^15654.940^20 782.747Appendix 11 ANOVA for the effect of CE on bud breakand RGP in Engelmann spruce seedlings.(A) Bud break at 268 days.SV^SS^DF^MS^F-RatioWater baths 24.942 2 12.471 8.592 0.000Treatments 18.724 5 3.745 2.580 0.031Water bath*treat. 14.162 10 1.416 0.976 0.470Error 139.348 96 1.452(B) Bud break at 327 days.SV SS DF MS F-Ratio PWater baths 1.975 2 0.987 0.095 0.909Treatments 95.740 5 19.148 1.847 0.111Water bath*treat. 78.016 10 7.802 0.753 0.673Sampling error 953.524 92 10.364(C) RGP at 268 days.SV SS DF MS F-Ratio PWater baths 18113.389 1 18113.389 17.726 0.000Treatments 5798.611 5 1159.722 1.135 0.352Water bath*treat. 3419.111 5 683.822 0.669 0.648Error 61310.667 60 1021.844(D) RGP at 327 days.SV^SS^DF^MS^F-Ratio^PWater baths 4383.865 2 2191.932 1.341 0.267Treatments 17850.197 5 3570.039 2.185 0.063Water bath*treat. 14856.357 10 1485.636 0.909 0.528Sampling error 145437.357 89 1634.128116Appendix 12. Protocol for Carbohydrate Analyses.I. Grinding the plant material (needles, roots and stems)Grind the freeze-dried plant material in liquid nitrogen usinga mortar and pestle. Needles are easily crushed by thismethod, but roots and stems may be difficult to grind and mayrequire milling. The ground material can be stored in vialsat 2 - 6°C until required for analysis. Tissues ground inliquid nitrogen pick up some condensation, and shouldtherefore be dried again prior to storage.II. Separating soluble sugars from starch1. Weigh out 20 to 50 mg of the pulverized plant material and putit in a test tube.2. To each test tube add 5 mL of methanol: choloroform: water(M:C:W - 12:5:3, v/v/v) and leave the samples overnight.3. The following morning, remove the samples from the freezer,briefly shake them on a vortex and then centrifuge for 10minutes (desk top). Pipet the supernatant containing solublesugars into vials and save. Starch will remain with thepellet.4. Add 3 mL of M:C:W to the pellet in the test tube. Vortexbriefly and then centrifuge for 10 minutes (desk top).5. Pipet off the supernatant and combine with the one from step3.6. Store the pellet in the freezer or take it immediately forstarch analysis.III. Final extraction of soluble sugars1. To each vial of supernatant from above, add 3 mL of distilledwater. Shake the vials gently by hand and let settle (about 5minutes).2. Pipet the top aqueous layer into an evaporating flask. Thelower layer is waste, mostly chloroform. Flash evaporate allthe solvent from the flask, while the flask rotates in a 40°Cwater bath. It takes 5 - 10 minutes to do one evaporation.3.^Add 1.5 mL of distilled water to the flask, swirl gently andpipet into a microcentrifuge tube. Wash the flask withanother 1.5 mL of distilled water, swirl gently and add to themicrocentrifuge tube. Spin for 3 minutes and transfer thesupernatant into vials and store solutions in the freezeruntil required for sugar analysis.117IV. Starch analysis using enzymes1. To the pellet in the test tube, add 5 mL of acetate buffer (pH4.5, 150 mM) and briefly vortex.2. Autoclave at 120°C for 1 hour or incubate in a water bath at88°C for 3 hours.3. Put the samples in a 55°C water bath for about 5 - 10 minutes.4. Add to each test tube:- 62.5 pL a-amylase solution (see VI.3)- 125 gL amyloglucosidase solution (see VI.2)Gently shake the contents by hand to mix the enzymes and thestarch solution. Incubate for 2 hours then cool for 5 - 10minutes at room temperature.5. Centrifuge for 10 minutes (desk top).6. Aiming for a working volume of 200 AL, pipet from step 5 thedesired amount of aliquot and dilute it appropriately. Thisrequires some sense of what the starch concentration in aparticular plant extract might be (i.e., varies with time ofyear, plant part, etc.). For spruce, the following areusually reasonable:Needles: 200 AL plant material + 0 AL acetate bufferRoots: 100 AL plant material + 100 pL acetate bufferStems: 150 AL plant material + 50 AL acetate buffer.7. Add 2 mL of combined peroxidase/glucose oxidase/colour reagentsolution.^See section VI.4 or refer to the attached SigmaDiagnostics Procedure No. 510.8. Incubate the samples for 30 minutes in a 37°C water bath.9.^Read the absorbance at 450 nm.Refer to: HAISSIG, B.F. and DICKSON, R.E. 1979, Physiol Plant47:151-157.Starch concentrations are calculated in glucose equivalents.When establishing a standard curve, first prepare a starchstandard solution by dissolving 16.2 mg of soluble starchpowder (BDH chemicals, ACS 879) in 100 mL of acetate buffer.The procedure in step IV was used to establish the standardcurve using the following quantities: 0.00, 50, 250, and 500nmol of glucose equivalents (Fig. 17b).V. Soluble sugars analysis using Anthrone Reagent1.^Take the supernatants from step III and dilute themappropriately. As in the starch analysis, the dilution factor118will vary from one plant part to another, time of year, etc.For spruce try the following:Needles: 40 AL plant extract + 160 AL distilled water.Roots: 50 gL plant extract + 150 ML distilled water.Stems: 60 gL plant extract + 140 pL distilled water.2. To deproteinize, add to each of the above 50 mL each of bariumhydroxide and zinc sulphate solutions (0.3 M).3. Centrifuge for 3 minutes, then pipet 200 AL of the supernatantinto a test tube.4.^To each of the above (step 3), add:- 200 AL of 11.6 M HC1- 40 AL of 45% formic acid- 1.6 mL of 80% sulphuric acid with anthrone^(i.e., 20 mganthrone (Sigma A-1631) plus 100 mL of 80% sulphuric acid).For each day prepare fresh samples of 80% H 2SO4 and anthronesolution.5.^Close the top of the test tube with a marble. Vortex veryvery gently and briefly (watch out for bubbles).6. Put the entire rack in a boiling water bath for exactly 12minutes.7. After 12 minutes, remove the rack and put it in an ice waterbath until cold (5 - 10 minutes).8. Vortex briefly. Let the samples sit in the ice water bath for5 - 10 minutes to let the bubbles settle.9. Read the absorbance at 630 nmol.Refer to: JERMYN, M.A. 1975. Anal Biochem 68:332-335.YEMM, E.W. and WILLIS, A.J. 1954. Biochem 57: 508-514.Glucose concentrations were calculated from a glucose standardcurve. Prepare the glucose standard solution by dissolving 18mg of D-glucose (BDH Inc. B10117) in 100 mL of distilledwater. The procedure in step V was used to establish thestandard curve using the following quantities: 0.00, 50, 100,150, and 200 nmol glucose (Fig. 17a).VI. Preparation of solutions used in the carbohydrate analyses1.^Acetate buffer (150 mM)a) Weigh out 4.082 g of sodium acetate powder into a measuringcylinder. Add distilled water to 200 mL level and stir well.119b) In a second cylinder, measure 2.58 mL of acetic acid. Thencarefully add distilled water to 300 mL level and stir well.c) Mix a) and b) together. The pH of the mixture should beapproximately 4.5.^2.^Amyloglucosidasea) Weigh out 800 units of amyloglucosidase powder(E.C.3.2.1.3, from Rhizopus, Sigma A-7255) and dissolve it in1.0 mL of distilled water.b) Add 3-5 mg of charcoal, mix very well and then spin (desktop).^Pipet the supernatant and to it add 0.4228 g ofgranular ammonium sulphate. Mix gently.c) Spin (desk top). Pipet the supernatant out and keep thepellet. Re-suspend the pellet in 1.0 mL of distilled waterand mix gently. If you get some charcoal particles, re-spinand save the supernatant.3.^a-Amylasea) Weigh out 40,000 units of a-amylase powder (E.C.3.2.1.1,from Aspergillus, Sigma A-0273) and dissolve it in 1.0 mL ofdistilled water.b) as in 2. b)c) as in 2. c)4.^Peroxidase/glucose oxidase/colour reagenta) EnzymesCapsules containing 500 units of glucose oxidase (fromAspergillus niger), 100 Purpurogallin units of peroxidase(horseradish) and buffer salts can be purchased from Sigma(PGO Enzymes, cat. no. 510-6). Dissolve 1 capsule in 100 mLof distilled water in an amber bottle. Mix well by invertingthe bottle several times.b) o - dianisidine dihydrochloride (colour reagent)Weigh out 50 mg of o-dianisidine dihydrochloride powder anddissolve it in 20 mL of distilled water. Mix well.c) To a) add 1.6 mL of the colour reagent solution from b) andmix well by inverting the bottle several times.For more details refer to Sigma Diagnostics Procedure No. 510.This leaflet accompanies kit 510-DA which comes with PGOEnzymes, colour reagent, glucose standard, and bariumhydroxide and zinc sulphate solutions.lie^atie^300^400GLUCOSE EQUIVALENTS (nnol)120(A)GLUCOSE STANDARD CURVE.S6^lee^150^200^250GLUCOSE (nmol)(B)STARCH STANDARD CURVE.Figure 17. Glucose (a), and starch (b) standard "curves"developed for carbohydrate analyses.BIOGRAPHICAL INFORMATION NAME: Bernard Malata ChombaMAILING ADDRESS: Forest Department, Division of Forest ResearchP.O. Box 22099, Kitwe. ZAMBIA.PLACE AND DATE OF BIRTH: Mporokoso District, Zambia, Dec. 05, 1953.EDUCATION (Colleges and Universities attended, dates, and degrees):University of Dar-es-Salaam, Morogoro, Tanzania: B.Sc. (nor.) (Hons.) 1982Zambia Forest College, Mwekera, Kitwe, Zambia: Dip. in Forestry, 1979POSITIONS HELD:Silviculturist/Fuelwood Project Manager: Jan. 1985 - Aug. 1989Silviculturist: Jan. 1983 - Dec. 1984Tree Improvement Forester: May - Dec. 1979PUBLICATIONS (if necessary, use a second sheet):(see attached sheet)AWARDS:1989 -1992: International Development Research Centre (IDRC, Ottawa, Canada)1986: British Council Scholarship, Oxford, U.K.1984: Finnish International Development Agency (FINNIDA, Helsinki, Finland)1980 - 1982: World Bank/Zambia Government Scholarship1977 - 1979: Zambian Government Forestry Diploma ScholarshipComplete one biographical form for each copy of a thesis presentedto the Special Collections Division, University Library.PUBLICATIONSChomba, B.M., and J. Saramaki. 1985. Coppice yield for shortrotation Eucalyptus on the Copperbelt. Zambia ForestDepartment. Research Note 37. 16 p.Chomba, B.M., and A.C. Mubita. 1988. Three year results of afuelwood research project in Zambia. Zambia ForestDepartment. Country Report. 52 p.Saramaki, J., and B.M. Chomba. 1985. Effects of pruning on thegrowth of Pinus kesiya. Zambia Forest Department.Research Note 35. 13 p.Selander, J., and B.M. Chomba. 1989. Control of Lantana camaraL. in forest plantations. Zambia Forest Department.Research Note 44. 12 p.Selander, J., and B.M. Chomba. 1989. Eradication of stumpregrowth in Eucalyptus grandis. Zambia Forest Department.Research Note 45. 11 p.

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