EFFECTS OF CARBON DIOXIDE AND DAYLENGTH ON GROWTH, DEVELOPMENT AND HARDINESS OF DOUGLAS FIR CAROLE LOUISE (SCHEUPLEIN) LEADEM

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1 EFFECTS OF CARBON DIOXIDE AND DAYLENGTH ON GROWTH, DEVELOPMENT AND HARDINESS OF DOUGLAS FIR (Pseudotsuga menziesii) by CAROLE LOUISE (SCHEUPLEIN) LEADEM B.Sc, University of California, Berkeley, California, 1973 A THESIS SUMBITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Faculty of Graduate Studies in the Department of Botany We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1979 CAROLE LOUISE LEADEM, 1979

2 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of BOTANY The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date 1 May 1979

3 ABSTRACT Seedlings and one-year-old trees of Douglas-fir (Pseyidotsuga menziesii) were grown in chambers with atmospheres maintained at 0.03% to 5.0% CO2 (by volume). Carbon dioxide treatments were given in conjunction with daylengths of 8 or 16 hours and light intensities -2-2 which varied from 3.4 mw cn to 7.2 mw cm ( nm). The duration of treatment varied from 30 days to 12 weeks. When plants were treated with 0.1% CX^ both seedlings and trees showed enhanced growth, as demonstrated by increases in dry weight and internodal elongation. C0 enrichment caused growth enhancement to a degree that other factors became limiting, e.g., irradiance. Carbon dioxide concentrations of 1.0% CC^ and higher generally inhibited growth, as shown by decreases in internodal elongation, dry weight, and leaf area. Plants grown under high carbon dioxide levels ceased active growth and exhibited increased budset and frost hardiness. High CO2 levels appeared to override photoperiodic control of budset by promoting budset even under warm temperatures and long days. COj-induced frost hardiness appears to require an active metabolism, indicating that the mode of CO2 action is through increased production of cryoprotectents, such as amino acids and sugars. ' '.' 1 ' ')I.'- ; In some cases, carbon dioxide may substitute in part for the light requirements of photosynthesis when light is limiting. Thus, an increase in daylength may reduce the level of COj required for a particular effect, e.g., the required C0 2 levels for inducing frost hardiness are reduced from 1.0% to 0.1% C0 9 if long days are

4 provided. Plants which have been grown under normal air (0.03% CO2) i i i have higher photosynthetic rates than enriched plants when all plants are measured in normal air; the concentration under which plants are measured appears to have more effect on photosynthetic rates than the CO2 concentration under which the plants are grown. Under long days and high CO2 (1.0% CO2 and higher), plants show reduced diffusion resistance, but the beneficial effects on photosynthesis due to potential reduction of CO2 diffusion resistance are lost as a result of increased rates of respiration under high CO2. Thus, the enhancement of growth under 0.1% Ct>2j and the inhibition of growth under 1.0% CO2, appear to be mostly related to differences in respiration under the various carbon dioxide treatments. All effects of carbon dioxide may not be due to gas exchange characteristics alone, but may result from changes in levels of growth inhibitors, such as abscisic acid. The effects of CO2 on growth and development were examined over a range of CO2 concentrations. Over the entire range CO 2 was found to effect both growth and development and the processes underlying growth and development.

5 TABLE OF CONTENTS Page ABSTRACT i i ACKNOWLEDGEMENTS x DEDICATION xii PREFACE xiii CHAPTER I. Effects of carbon dioxide and daylength on growth of Douglas-fir (Pseudotsuga menziesii) INTRODUCTION. 1 MATERIALS AND METHODS 5 RESULTS A. HEIGHT 1. One-year-old trees (a) Effects of 8- and 16-hour daylengths and two carbon dioxide levels (b) Effects of 16-hour daylength and three carbon dioxide levels Six-month-old seedlings: experimental design (a) Effects of daylength 16 (b) Effects of carbon dioxide 16 (c) Effects of carbon dioxide and daylength.. 19 (d) Effects of temperature Summary: Effects of carbon dioxide and daylength on height 23 B. WEIGHT 1. One-year-old trees (a) Effects of 8- and 16-hour daylengths and two carbon dioxide levels 25 (b) Effects of 16-hour daylength and three carbon dioxide levels (c) Effects of 8-hour days and four carbon dioxide concentrations Six-month-old seedlings Summary: Effects of carbon dioxide and daylength on weight (a) Leaves 41 (b) Stems 42 (c) Roots 42 (d) Biomass Distribution 42 DISCUSSION 44

6 CHAPTER II. Effects of carbon dioxide and daylength on dormancy and hardiness of Douglas-fir INTRODUCTION... MATERIALS AND METHODS RESULTS V Page FLUSHING AND BUDSET 1. One-year-old trees (a) Effects of 8- and 16-hour daylengths and two carbon dioxide levels. 67 (b) Effects of 16-hour daylengths and three carbon dioxide levels (c) Effects of 8-hour daylength and four carbon dioxide levels Six-month-old seedlings (a) General flushing and budset behaviour (b) Maximum budset 75 (c) Earliest date of budset 75 (d) Effects of temperature on plants grown under air (0.03% C0 2 ) 79 B. COLD HARDINESS 1. Effects of carbon dioxide (a) Preliminary study 79 (b) Plants grown under 8-hour days 82 (c) Plants grown under 16-hour days Effects of post-treatment (a) Plants grown under 8-hour days 86 (b) Plants grown under 16-hour days Effects of temperature Effects of C02 and daylength on amino acids '. 92 DISCUSSION 95 CHAPTER III. Effects of carbon dioxide and daylength on gas exchange of Douglas fir INTRODUCTION 110 MATERIALS AND METHODS 112 RESULTS 116 A. PHOTOSYNTHESIS 1. Plants grown under 8-hour days and four CO2 levels

7 2. Plants grown under 16-hour days and four levels B. RESPIRATION C. TRANSPIRATION 1. One-year-old trees 2. Six-month-old seedlings D. TOTAL DIFFUSIVE RESISTANCE DISCUSSION CONCLUSIONS LITERATURE CITED.. APPENDICES CO- vi Page,119 ' TABLE A TABLE B TABLE C Summary of Experiments # -,57 Ranking Codes 158 Schematic Diagram for Experiment Number TABLE D TABLE E Spectral Distribution of Sherer Growth Cabinet. Spectral Distribution of Mercury Vapor Lamps

8 LIST OF TABLES vii Table Page 1 Growth of trees grown under 8- and 16-hour photoperiods with either 0.03% or 1.0% CO Growth of trees grown under 16-hour photoperiods with either 0.03%, 0.1%, or 1.0% C Growth of seedlings. Effect of daylength 17" 4 Growth of seedlings. Effect of carbon dioxide 18 5 Growth of seedlings. Effect of carbon dioxide and daylength Growth of seedlings. Effect of temperature 24 7 Dry weight of trees grown under 8- and 16-hour photoperiods with either 0.03% or 1.0% C Shoot:root of trees grown under 8- and 16-hour photoperiods with either 0.03% or 1.0% C Shoot:root of trees grown under 16-hour photoperiods with either 0.03%, 0.1%, or 1.0% C Dry weight of trees grown under 16-hour photoperiods with either 0.03%, 0.1%, or 1.0% C Needle dry weight and area of trees grown under 8-hour photoperiods with 0.03%, 0.1%, 1.0%, or 5.0% C Regression of leaf area and leaf weight of trees Fresh weight of seedlings grown under 8- and 16-b.our daylengths and 0.03%, 0.1%, 1.0%, or 5.0% C Proportional weight distribution of seedlings Degree of lateral budset in trees grown under 8- or 16-hour daylengths with 0.03% or 1.0% C Degree of budset in trees grown under 16-hour daylengths with 0.03%, 0.1%, or 1.0% C Degree of budset in trees grown under 8-hour daylengths with 0.03%, 0.1%, 1.0%, or 5.0% C Date of first budset in trees grown under 8-hour daylengths with 0.03%, 0.1%, 1.0%, or 5.0% C0 0.73

9 Table Pag 19 Maximum number of buds set in seedlings grown under 8- or 16-hour daylengths with 0.03%, 0.1%, 1.0%, or 5.0% C Earliest date of budset in seedlings grown under 8- or 16-hour daylengths with 0.03%, 0.1%, 1.0%, or 5.0% C Earliest date of budset in seedlings grown under 8-hour daylengths with three temperatures Frost hardiness study of trees Cold hardiness of seedlings. Effect of carbon dioxide Cold hardiness of seedlings grown under 16-hour days. Effect of carbon dioxide Cold hardiness of seedlings grown under 8-hour days with 0.03%, 0.1%, 1.0%, or 5.0% C0 2> Effect of post-treatment Cold hardiness of seedlings grown under 16-hour days with %, 0.1%, 1.0%, or 5.0% C0 2. Effect of post-treatment.. 90 i Cold hardiness of seedlings grown under 8-hour daylengths with three temperatures. Effect of temperature Amino acids in leaf extracts of 1-year-old trees Rates of photosynthesis of seedlings grown under 8-hour days Rates of photosynthesis of seedlings grown under 16-hour days Rates of transpiration of seedlings grown under 8-hour days Rates of transpiration of seedlings grown under 16-hour days. 130

10 LIST OF FIGURES ix Figure Rage 1 Height increases after treatment with C^-enriched atmospheres (1-year-old Douglas-fir trees) 11 2 Height increases under 16-hr days (1-year-old Douglas-fir trees) ^ 3 Effect of CO2 on height of 6-month-old seedlings 22 4 Shoot fresh weight increases (1-year-old Douglas-fir trees) Root fresh weight increases (1-year-old Douglas-fir trees) Degree of budset under 8-hr days (Douglas-fir 6-month-old seedlings) Degree of budset under 16-hr days (Douglas-fir 6-month-old seedlings) Degree of budset under 8-hr days and 3 temperatures (Douglasfir 6-month-old seedlings) 80 9 Photosynthetic rates of seedlings grown under 8-hr days Photosynthetic rates of seedlings gronw under 16-hr days Effect of daylength and CO2 on photosynthesis Effect of daylength and on respiration Weekly water loss (1-year-old Douglas-fir grown under 8-hr days and 4 C0 2 levels) 14 Transpiration rates of seedlings grown under 8-hr days Transpiration rates of seedlings grown under 16-hr days.. 16 Effect of daylength and CO2 on total resistance

11 ACKNOWLEDGEMENTS I would like to thank my main advisor, Tony Glass, for all the work he has done on my behalf in the Department. Since I was an off-campus student he took care of many tasks which I would have found very difficult to do without his help. I sincerely appreciate all of his efforts to bring this work to completion. I also wish to give my thanks and appreciation to Peter Jolliffe who so kindly took over as my advisor after the death of Bruce Tregunna. His guidance during the later part of my research and his many suggestions during the writing of this thesis were invaluable in completing my work. I am very grateful to Pawan Bassi, with whom I shared the laboratory. His good humor and encouragement helped me over many of the hard times. The following people are only a few of those many kind friends and associates who gave me assistance during my research, but my thanks are extended to all those whom I did not have room to mention specifically. I would like to thank: Geoff Lister, who advised me on various aspects of gas exchange and who graciously allowed me to examine his unpublished data on western hemlock. the members of my committee: Andy Black, who taught me not to fear mathematical equations; and Ron Foreman, who always found time to give me help and advice. Mel Davies, the Botany Department Saviour of Machines and Equipment, who nursed my ailing mechanical monsters countless times.

12 xi Our discussions, both scientific and philosophical, contributed greatly to my education during my four years at U.B.C. my children, John and Lauryn, for their moral support and for their assistance in the laboratory, even though they could never understand why Mom went to school when she didn't have to. Finally, I would like to thank Tim whose love and moral support kept me going, and whose tangible support assisting me in the laboratory, taking photographs, drafting figures, critically reading and re-reading this manuscript helped me finish. For their contributions of Douglas fir plants used in this research I would like to thank the B.C. Forest Service and Weyerhaeuser Company, Centralia, Washington.

13 DEDICATION xii This thesis is dedicated to the memory of E. Bruce Tregunna, scientist, teacher, and friend. Bruce Tregunna was a truly creative, open-minded scientist, and was worthy of emulation. Bruce created a supportive atmosphere in his laboratory and was largely responsible for the co-operative interaction of all members of his group. He promoted the exchange of ideas from researchers outside his laboratory as well, and whenever visitors came to campus we would all gather in the coffee room for informal discussions. These discussions were a valuable educational experience which served to broaden both my intellectual and scientific perspectives. As my major advisor, Bruce was at all times enthusiastic and encouraging. His support was invaluable to me during the progress of this research. In our personal discussions, Bruce was an attentive listener and receptive to new ideas. He provided guidance when needed but he also allowed me the maximum amount of freedom for research and problem-solving. In addition, Bruce was a compassionate and thoughtful human being for whom I had the greatest admiration. The thought of his death shall always inspire me with a sense of loss.

14 xiii PREFACE In the laboratory of E. Bruce Tregunna, work on the effects of carbon dioxide enrichment was just beginning when I began my graduate studies. Bruce Tregunna and Aditya Purhoit were screening plants for their response to CO^. and as part of this work, they grew Douglas fir seedlings under high CO^ levels. After 90 days they examined the plants and found that several plants grown under 1.0% CO,, had formed curious clusters of bract-like leaves which resembled the early developmental stages of female cones. This finding was quite different from the usually observed effects of favourable levels of CO2 enrichment, i.e., the enhancement of dry weight and internodal elongation. It appeared that carbon dioxide which is a relatively simple, molecule basic to plant metabolism could affect plant growth and development in ways other than providing essential nutrition. Intrigued by these preliminary results I became interested in further exploring carbon dioxide effects on growth and development of Douglas fir. Other members of our research team investigated other carbon dioxide effects on physiological events, such as the interaction of C0 2 and phytochrome. I was able to only sporadically reproduce the effects of C0 2 on bud development which originally had caught my interest in this research however, other effects of C0 2 which I observed during the course of my investigations turned out to be equally as interesting. This thesis includes experiments relating to several different effects of carbon dioxide on growth and development of Douglas fir. The experiments were designed to define the ranges in which C0 2 enhances growth and the ranges in which C0 2 inhibits growth,

15 as well as explore the interactions between CO^ and other factors xiv influencing growth. The effects of CO^ on budset and on freezing resistance were also studied. An attempt was made to determine the relationships between growth, degree of budset, and freezing resistance. In addition, I investigated the effects of carbon dioxide on photosynthesis, respiration, and transpriation and made an effort to determine the interrelationships between the different components of gas exchange.

16 1 CHAPTER ONE EFFECTS OF CARBON DIOXIDE AND DAYLENGTH ON GROWTH OF DOUGLAS FIR (Pseudotsuga menziesii) INTRODUCTION Investigation of the effects of carbon dioxide enrichment on plant growth began about the turn of this century. As early as 1903 there were reports of dry weight increases of as much as 158% when carbon dioxide was added to the plant atmosphere (48). Much uncertainty surrounded this early work because other workers reported negative effects of enrichment, such as reduction in leaf area, slow development of internodes, and retardation of flower and fruit development. In retrospect, not all of these problems may have been due to CO^, but to other factors, such as contaminants in the gases used for enrichment, and excessive humidity in the treatment chambers. Conflicting reports may have also been due to difficulties with accurately measuring the CO^ concentrations around the plant. It was not then known that specific plants may exhibit a fairly well-defined optimum range for carbon dioxide, and that inhibitory effects can quickly increase once the optimum is exceeded. Work in the field continued, however, until about 1930 when it gradually declined. Interest in CO^ enrichment was revived when in 1964 Wittwer and Robb (85) detailed the many advantages that horticulturists could achieve by using CO^ enrichment in the greenhouse atmosphere. In cucumber, there were more pistillate flowers which resulted in a 72% increase in fruit (85) and in petunias, accelerations in flower development (38). Lettuce is an ideal crop for enrichment, as CO^ promotes increases in both leaf size and thickness, plus

17 2 accelerating growth, making it possible to grow four crops in one year rather than just three (83). Benefits from enrichment are realized in many fruits, with improvements in fruit color and fruit shape, and reduction in the number of scars (37). Outside the horticultural field, only limited work with carbon dioxide enrichment has been done, but of special interest is work relating to enhancing the growth of trees. Carbon dioxide has been found to improve rooting of softwood, conifer, and herbaceous cuttings (50). Levels of 1000 ppm CO^ doubled height and growth of white pine seedlings (22), while levels of 900 and 1500 ppm CO^ increased dry weight 30% to 80% in 3-week-old seedlings of white spruce, Norway spruce, jack pine and Scots pine (86). Other reports show positive CO^ effects on the growth of Douglas fir, western hemlock, noble fir, white fir, ponderosa pine, and lodgepole pine (E. B. Tregunna, unpublished data, 1974; 71, 72), but the potential benefits of applying carbon dioxide enrichment to economically important trees are far from being realized. To my knowledge there have been few studies on optimum levels for tree growth enrichment, and no exploration of levels at which carbon dioxide becomes inhibitory to growth. The highest reported levels of CO2 used in tree growth studies have not exceeded 1500 ppm CO^ (71, 72). Daylength also has important implications relating to seasonal and annual cycles of growth, and to the development of perennial species which undergo alternating periods of active growth and dormancy. There has been some work done in the past on the effects of different light

18 3 intensities in conjuction with several levels of carbon dioxide, but no work has been reported on the effects of different daylengths combined with several carbon dioxide levels. The broad objective of the research presented in this chapter was to determine how carbon dioxide and daylength influenced the growth processes of Douglas fir. I attempted to answer the following questions: What concentrations of CO^ promoted growth, and what concentrations inhibited growth? within the plant? Did carbon dioxide affect the biomass distribution What were the effects of light on growth, and did irradiance levels or photoperiod affect the expression of the CO^ effect? Did CC^ interact with light or any other factors to influence growth? Finally, could the effects of CO,, persist after enrichment terminated? To accomplish my research objective I conducted a series of experiments. Each experiment was designed to contribute a part to the cumulative knowledge required to answer the questions above. Initially I wished to determine the general ranges of daylength and carbon dioxide which would promote growth of 1-year-old Douglas fir trees. Two different photoperiods (8 and 16 hours) and two different carbon dioxide levels were used for the first experiment (0.03% and 1.0% CO,,). The plants were grown for only 30 days, but some general observations on the effects of CO,, and daylength could be made. The observations were used to plan a second experiment in which both the duration and the number of CO,, levels were increased; thus plants were grown for 90 days under three C0 2 levels (0.03%, 0.1% and 1.0% C0 2 ). The dual objectives were to

19 4 observe the long-term effects of CO,, on height and weight, and to determine the best CO^ level for enhanced plant growth. Together the results of experiments 1 and 2 provided enough data to determine the ranges in which CO,, promoted growth, but only limited data on the point at which (X^ would begin to inhibit growth. Accordingly, in experiment 3 a higher CO,, level (5.0% CO,,) was added to the existing three C0 2 treatments. Since light levels had previously been limiting plant response, irradiance was also increased. The cumulative data gathered from the results of the first three experiments presented a general picture of the response of 1- year-old Douglas fir trees to CO,, and daylength, however, I had little knowledge of how CO^ daylength affected plants younger than one year. There were several reasons for being interested in the effects of CO^ on young plants. CO,, enrichment during the seedling stage probably would produce the greatest growth benefits since young plants generally respond more readily to environmental changes, and have greater relative growth rates. Smaller plants are easier to handle because of their size, and fit more easily into controlled environment chambers. Using younger plant material would also make it possible to increase sample sizes, consequently reducing total variability within the sample. Finally, seedling research has good potential for practical benefit in British Columbia where forest nurseries require large numbers of trees each year. Reforestation nursery programs are always seeking new techniques for accelerating seedling growth in order to produce plantable trees in the shortest possible time.

20 5 With the above considerations in mind, 6-month-old seedlings were grown for six weeks with either 8- or 16-hour photoperiods under four different CC^ levels, then transferred to normal air for another four weeks (Appendix, Table A, Exp. No. 5). The first part of the final experiment provided data on the general effects of CC^ and daylength on seedling growth, whereas the second part provided additional data on the persistence of CO^ effects after the plants were grown in normal air. The completion of the experiments described in this chapter supplied most of the results needed to determine how carbon dioxide and daylength influenced some of the growth processes of Douglas fir. Most of my initial questions were answered although some of the results raised even more questions. Regardless, the cumulative data did enable me at least to formulate a tentative picture of CC^ effects and its interactions with other factors influencing growth. MATERIALS AND METHODS Plant Material One-year-old Douglas fir trees (1-0 stock) were obtained from the British Columbia Forest Service from either their South Surrey or Green Timbers bare root nursery. The coastal provenance trees had been grown from seeds originally gathered at an elevation of approximately 500 m on the east coast of Vancouver Island. Trees were grown in 15 cm pots filled with Mica-Peat (a sterilized peat moss and vermiculite mixture) obtained from Langley Peat Limited, Fort Langley, B.C. Seedlings

21 6 were supplied by Weyerhaeuser Company, Centralia, Washington, and were also coastal provenance Douglas fir from Twin Harbours, Aberdeen, Washington (elevation 300 m t 60 m). The plants had been seeded into individual flexible plastic plugs filled with a standard potting mixture. The seedlings were not transplanted since the plugs were of sufficient volume to allow for reasonable root expansion. Both trees and seedlings were well-watered and fertilized every two weeks with Hi-Sol (N-P-K) solution. Terminology To simplify the terminology throughout this thesis, CO^ concentrations cf 0.03% CO^ (v/v) will be referred to as either "atmospheric", "normal", or "low" C0 2 levels; 0.1% CC> 2 will be called "intermediate" C0 2 levels; and 1.0% or 5.0% CC> 2 will be called "high" C0 2 levels. Exact concentrations will be stated in instances when descriptive labels might otherwise be misleading or ambiguous. Days with light periods of 8 hours will be called "short days" and those with 16 hours will be called "long days". Experimental Design A general summary of all experiments, given in the Appendix, Table A, can be used for reference when needed. The basic purpose of Experiment 1 was to compare the effects on growth of normal and enriched C0 2 levels under two different photoperiods. One-year-old Douglas fir trees were grown under 8- and 16-hour daylengths with either 0.03% or 1.0% C0 2 in chambers maintained

22 7 25 C/20 C. Experimental plants were screened to determine that initially all trees were of uniform size. Initial height and weight measurements were taken at the beginning of treatment and again after 7, 14, 21, and 30 days of treatment. Height measurements in this experiment and all others were taken from the root collar to the tip of the main stem. Dry weights were determined after plant material had been dried at 85 C for 48 hours. In Experiment 2, the number of CO^ levels was expanded to determine the effects of varying concentrations, and to establish the optimum concentration for growth enhancement. Trees were grown under 0.03%, 0.1%, and 1.0% CO^. hut photoperiod (16-hour days) was kept constant. In addition, the duration of the experiment was extended to 90 days to assess long-range growth patterns and responses. Height and weight samples were taken at the beginning of treatment and again after 15, 30, 60, and 90 days. The number of CO levels was again expanded in Experiment 3, but in this experiment the purpose was to ascertain the point at which would begin to inhibit growth. The focus of the experiment was on growth inhibition, therefore short photoperiods (8 hours) were chosen in conjunction with CO^ concentrations of either 0.03%, 0.1%, 1.0%, or 5.0% After treatment for 21 days, needles from each of the four treatments were sampled and dry weights and areas were determined. Regressions based upon the data were also performed for each of the four treatments.

23 8 The overall objective of the final experiment was to study the growth of 6-month-old Douglas fir trees (Appendix, Table A, Exp. No.5). There were two main parts to the experiment, a pretreatment and a post-treatment (see schematic, Appendix, Table C). The purpose of the first part was to determine the effects of CO^ and photoperiod on growth, and consisted of pretreating plants for six weeks with either 8- or 16-hour days (25 C/20 C) and one of four C0 2 levels (0.03%, 0.1%, 1.0%, or 5.0% C0 2 ). During the second part of the experiment, all plants were transferred to normal air and maintained under short warm days (25 C) and long cold nights (5 C) for four weeks. Additional controls were grown continuously under short days and normal air, but given thermoperiods of either 25 C/5 C or 5 C/5 C. (The first number given is the temperature during the light period, while the second number is the temperature during the dark period). The primary purpose of the post-treatment was to establish whether the effects of CC^ pre-treatment persisted beyond the actual CO^ enrichment period, but there was a secondary purpose as well. In nature, short warm days and long cold nights promote the cessation of growth and the onset of dormancy. Many of the plants had already been pre-treated under conditions which enhanced growth, however seedlings were also to be post-treated with conditions which induced dormancy. Thus, the secondary objective of the post-treatment was to ascertain whether pre-treatment or whether post-treatment would exert the greatest influence in controlling plant growth response. For this experiment height and weight samples were taken after six weeks of pre-treatment and after four weeks of post-treatment.

24 9 Growth Chambers Experiments 1, 2 and 4 (as listed in Table A, Appendix) were conducted in Sherer growth cabinets sealed to maintain ambient CO^ concentrations at the required levels. Cabinets were illuminated with mixed incandescent and flourescent lights. Radiant energy at midplant level in the photosynthetically active region ( mm) was mw/cm as measured by the spectral radiometer described by Burr and Duncan (89). A sample spectral distribution curve appears in the Appendix, Table D. Carbon dioxide was supplied from cylinders (Liquid Carbonic or Matheson Supply Company) and a Matheson Rotameter (a precision flowmeter with an adjustable valve) regulated the flow. Gas within the chamber was monitored periodically with a Beckman IR-215 infrared gas analyzer. Experiments 3 and 5 were conducted in eight specially constructed Plexiglas chambers (59). A thermistor monitored air temperature and a cooling coil provided humidity control within each chamber. Condensed water collected at the base of the cooling coil and drained to the outside of the chamber through Tygon tubing to another collecting vessel. A small fan mounted on the Plexiglas wall assured that air within each chamber was well mixed. A thermostatically cooled circulating water bath maintained at a depth of 7 cm reduced infrared radiation from the two 1000 W mercury vapour flourescent lamps (Duroglo GA 217G2) mounted above the eight chambers. Carbon dioxide from cylinders mixed with air pumped from outside the laboratory provided the gas supply to the chambers. Air from each of the eight chambers was

25 10- automatically sampled in turn using an aquarium pump inside each chamber; pumped gas was directed to a Beckman Model IR-215 infrared gas analyzer. Solenoid valves were activated whenever the millivolt output from the gas analyzer fell below the set point, injecting additional CO^ into the chamber, and thus providing a constant CO^ level. There were occasional problems maintaining CC^ levels due to failures in equipment however, CC^ concentrations were checked at least daily and usually several times a day and adjustments were made as necessary. Occasional fluctuations of CO^ levels would probably have minimum effect on the overall results of long-term experiments lasting a month or more. In any case, CO^ fluctuations would be of importance primarily in explaining th'e lack of results due to carbon dioxide enrichment, and not for explaining the positive effects of C0. RESULTS A. HEIGHT 1. One-year-old trees (a) Effects of 8- and 16-hour daylengths and 2 CO,., levels on height Initially, plants in all four treatments were approximately the same height, but by the third week significant differences between the treatments were apparent (Fig 1, Table I). Trees continuously maintained under atmospheric C0 0 and long days (16 hours) were noticeably

26 Figure 1. Mean cumulative elongation for 1-year-old Douglas fir continuously supplied with C02 at the levels shown, and maintained under either 8- or 16-hour photoperiods (25 C/20 C) with an irradiance of 3.4 mw cm b - 02% C02 LD % C02 SO 1.0% co2 SO 1.0% C02 LD ~5.0 E u LU 24-0 o 63.0 Ul I IL^v!^ fr" 7 8 = I cn - Q t. i - s TIME (weeks) d / ys d 3 t a l n t S S W n l n t P h Analysis of Variance was used for determining statistical h e f u r e a r e t i& differences. m e^s of 8 plants. Values bearing the h e same letter within the same column are not significantly different at p =

27 12 TABLE I CUMULATIVE GROWTH INCREASE OF CO -ENRICHED DOUGLAS FIR TREES (1-0 Stock) Grown for 30 days under 8- and 16-hour photoperiods (25 C/20 C) with either 0.03% or l.d% C0 2 Irradiance = 3.4 mw cm -2 TREATMENT MEAN LENGTH OF NEW GROWTH (cm ) Daylength CO cone. 7 Days 14 Days 21 Days 28 Days (h) lo) a 5.05 a ab a 5.01 a 5.79 ab a 4.56 a 6.04 b 6.41 b a 3.86 a 5.08 a 5.50 a The data points shown in the table are the means of 8 plants. Analysis of variance was used for determining statistical differences. Values bearing the same letter within the same column are not significantly different from each other at p = 0.05.

28 13 taller than trees grown under the other three conditions. In contrast, trees grown under high CC^ and long days were the shortest of all four treatments. Atmospheres containing high CC^ tended to inhibit stem growth under both short and long days, but the differences were only statistically significant under long days. (b) Effects of 16-hour daylength and 3 C0 levels on height Significantly different trends between high and low CO,, plants were observable by 30 days (Figure 2, Table II). After that time, extension growth levelled off in both the low and intermediate groups, but around 60 days they resumed growth. Plants grown under high CO^ grew slower than the other two groups but continued at a fairly constant rate. Trees supplied with intermediate CO^ levels were the tallest at the end of 90 days, although they were relatively late in responding to CQ^ enrichment. High CO 2 levels appeared to inhibit elongation since the shortest trees were those grown under 1.0% CO2. Trees grown under atmospheric conditions were intermediate in height between the other two groups. In comparing Experiments 1 and 2, growth trends for the first 30 days were similar. In Experiment 1 the greatest differences between trees grown under normal and high CO2 levels occurred at three weeks, whereas in Experiment 2 the greatest differences between normal and high CO2 levels occurred at four weeks. Trees grown in normal air showed exponential and plateau phases in both studies, although the

29 Figure 2. Mean cumulative elongation of 1-year-old Douglas fir continuously supplied with CO at the levels shown, and maintained under 16-hour photoperiods (25 C/20 C) with an irradiance of 3.4 mw cm TIME (days) The data points shown in the figure are the means of 5 plants. Analysis of Variance was used for determining statistical differences. Values bearing the same letter within the same column are not significantly different at p = 0.05.

30 15 TABLE II CUMULATIVE GROWTH INCREASE OF CO -ENRICHED DOUGLAS FIR TREES (1-0 Stock) Grown for 90 days under 16-hour photoperiods (25r.C/20 C) with either 0.03%, 0.1% or 1.0% CO Irradiance = 3.4 mw cm TREATMENT MEAN LENGTH OF NEW GROWTH (cm ) 15 Days 30 Days 60 Days 90 Days 0.03% co a 6.92 b 8.08 a ab 0.1% co a 6.04 ab 7.14 a b 1.0% co a 4.62 a 6.38 a 8.60 a The data points shown in the table are the means of 5 plants. Analysis of Variance was used for determining statistical differences Values bearing the same letter within the same column are not significantly different from each other at p = 0.05.

31 16 pattern was more clearly discernable in the 90-day study. Trees in high increased at a lower rate, but grew continuously, and thus did not demonstrate a marked plateau phase. 2. Six-month-old seedlings (a) Effects of daylength on height Plant heights under all CO^ levels were averaged, then analyzed according to the daylength under which they had been grown (Table III). After six weeks with warm day and night temperatures (25 C/20 C), new growth of seedlings maintained under short days was not significantly different from that of seedlings grown under long days. The effects of long days were not apparent until the plants were transferred to air and given short warm days' and long cold nights which in nature would ordinarily promote growth cessation and dormancy onset in Douglas fir. In this instance, however, plants previously grown under long days continued to grow even in noninducing circumstances. (b) Effects of C0 on height Individual data points for all plants were averaged, then analyzed on the basis of their growth regime, regardless of the photoperiod they had received. Data analyzed in this manner demonstrated that CO^ effects could be seen both during the pre-treatment and posttreatment periods (Table IV). Seedlings given intermediate CO^ levels exhibited the best growth during pre-treatment and post-treatment and

32 17 TABLE III CUMULATIVE GROWTH INCREASE OF CO^ENRICHED DOUGLAS FIR SEEDLINGS A. EFFECT OF DAYLENGTH Irradiance = 7.2mW cm TREATMENT MEAN LENGTH OF NEW GROWTH (cm ) Daylength (25/20 C) 8-hr day for 6 weeks and (25/5 C) TOTAL for 4 wks 8-hour 0.98 a 0.08 a 1.06 a 16-hour 0.82 a 0.86 b 1.68 b The data points shown in the table are the means of 48 plants. Analysis of Variance was used for determining statistical differences. Values bearing the same letter within the same column are not significantly different from each other at p = Mean cumulative elongation of 6-month-old Douglas fir averaged for all C0 2 levels under each daylength. Plants were pre-treated for six weeks under 8-and 16-hour days (25 C/20 C) and four C02 levels (0.03%, 0.1%, 1.0%, and 5.0% CO2). At the end of pre-treatment plants were transferred to a four week post-treatment of 8-hour days and normal air (0.03% CO2) (25 C/5 C).

33 18 TABLE IV CUMULATIVE GROWTH INCREASE OF C0 2 ~ENRICHED DOUGLAS FIR SEEDLINGS B. EFFECT OF CARBON DIOXIDE -2 Irradiance = 7.2 mw cm TREATMENT MEAN LENGTH OF NEW GROWTH (cm ) Averaged for both daylengths (25/20 C) for 6 weeks a 8 - h r d a y s f <fc) for 4 wks. TOTAL 0.03% C a 0.59 a 1.34 a 0.1% C b 0.98 b 2.27 b 1.0% C a 5.0% C a 0.62 a 1.27 a The data points shown in the table are the means of 60 plants. Analysis of Variance was used for determining statistical differences. Values bearing the same letter within the same column are not significantly different from each other at p = Mean cumulative elongation of 6-month-old Douglas fir averaged for 8 and 16-hour daylengths under each C02 level. Plants were pre-treated for six weeks under 8- and 16-hour days (25 C/20 C) and four C02 levels (0.03%, 0.1%, 1.0%, and 5.0% CO2). At the end of pre-treatment plants were transferred to a four week post-treatment of 8-hour days and normal air (0.03% CO2) (25 C/5 C).

34 19 were significantly different from plants grown under low and high CO levels. These results were similar to those obtained for 1-year-old Douglas fir (see Figure 2). (c) Effects of CO,, and daylength on height The combined effects of photoperiod and carbon dioxide should be considered, since the separate effects of these two parameters have already been examined (Table V). Long days and intermediate CO,, levels were most effective in promoting growth during the pre-treatment period (i.e., the initial six weeks). However, while the mean height increase for seedlings grown under 16-hour days and 0.1% CO^ was greater than the means of all other groups, it was not significantly different from the means of shortday plants grown under low or intermediate CO,, levels. At the end of post-treatment (during which seedlings v/ere grown in normal air) three statistically different groups could be distinguished. The smallest growth increments occurred in the first group, consisting of plants pre-treated with short days. No differences in the short-day group could be attributable to CO., pre-treatment, but under long days responses to carbon dioxide enrichment could be found. Moderate growth occurred in the second group and was shown by those plants pre-treated with long days and either low or high C0 2 levels. The greatest growth occurred in the third group and was exhibited by plants pre-treated with long days and intermediate C0 2 levels. Plants pre-treated with long days and intermediate C0 2 also demonstrated the greatest total growth during the 12 week period, and were significantly different from all other treatment groups.

35 20 TABLE V CUMULATIVE GROWTH INCREASE OF CO^ENRICHED DOUGLAS FIR SEEDLINGS C. EFFECT OF CARBON DIOXIDE AND DAYLENGTH _2 Irradiance = 7.2 mw cm TREATMENT MEAN LENGTH OF NEW GROWTH (cm ) Daylength (h) COo cone. (%) (25/20 C) for 6 weeks 8-hr days and (25/5 C) for 4 wks TOTAL C 0.03 a 1.02 a c a ab 0.14 a 0.92 a ab 0.10 a 0.91 a b b 1.42 a C 1.24 C 2.54 b b 1.40 a b 1.34 a The data points shown in the table are the means of 15 plants. Analysis of Variance was used for determining statistical differences. Values bearing the same letter within the same column are not significantly different from each other at p = Plants were pre-treated for six weeks under 8- and 16-hour days (25 C/20 C) and four C0 2 levels (0.03%, 0.1%, 1.0%, and 5.0% CO2). At the end of pre-treatment plants were transferred to a four week post-treatment of 8-hour days and normal air (0.03% C0 2 ) (25 C/5 C).

36 2 1 General growth responses were the same for short-day and longday plants when height increases were plotted as a function of CO^ pre-treatment (Figure 3). Both groups showed a base value at low CO^ levels, an optimum at intermediate CC^ levels, and a decline at high CC>2 levels. The total growth of long-day plants was substantially greater than that of short-day plants, and the magnitude of the peak value was both larger and more clearly defined. There were no discernable differences between short-day and long-day plants after six weeks for 6 month-old Douglas fir, as had been observed for 1-year-old trees. The absence of notable differences between short- and long-day plants may have been due to experimental restrictions regarding use of the conditioning chambers. Plants to be used for the long-day portion of the experiments had to be stored under long days in the greenhouse while short-day plants were being treated in the experimental chambers. While under greenhouse conditions the plants continued to flush, which probably made them less responsive to further treatment. It should be noted, however, that when the long-day seedlings were post-treated under conditions which normally do not promote growth (short warm days and cold nights), long-day plants showed greater total overall growth than short-day plants. It appears, therefore, the effects of long photoperiods can persist, extending the period of growth enhancement beyond the time of actual treatment.

37 22 Figure 3. Mean cumulative elongation of 6-ironth-old Douglas fir continuously supplied with CO2 at the levels shown, and maintained under either 8- or 16-hour photoperiods with an irradiance of 7.2mW cm-2, then post-treated with 8-h days (25 C/5 C) for 4 weeks. Increase in height after 6 weeks of CO2 pretreatment plus 4 weeks of post-treatment (8-hour days with 25 C/5 C). The data points shown in the figure are the means of 15 plants. Analysis of Variance was used for determining statistical differences. Values bearing the same letter of the same case are not significantly different from each other at p = 0.05.

38 23 (d) Effects of temperature on height Control plants were grown under atmospheric CC^ levels and three different thermoperiods to examine temperature effects upon growth (Table VI). Plants with warm days and nights grew nearly twice as much as those grown with cooler temperatures. There was no difference noted between plants grown with warm days and cold nights, and those grown with cold days and nights. All these groups of plants were maintained under short days; they responded similarly to the shortday plants discussed previously (in Section c); i.e., most growth occurred during the first six weeks and little growth occurred during posttreatment. 3. Summary: Effects of CO,, and daylength on height Carbon dioxide had similar effects on height when the same CO -daylength combinations were used on 1-year-old and 6-monthold plants. In some cases the data were variable, and although plants given low levels of enrichment might have had the highest mean values, the means could not be shown always to be statistically different from the means of other treatments. Generally however, low levels of enrichment (0.03% and 0.1% CO^) promoted internodal elongation, whereas high levels of enrichment (1.0% and 5.0% CO^) inhibited elongation. In experiment 1, long-day plants showed the greatest elongation under low CO^ and the least elongation under high C02«Also in experiment 3, plants grown under low enrichment levels (0.1% CO^) were the tallest after 90 days, while those under high enrichment levels (1.0% CO,,) were the shortest. The main difference between the results

39 24 TABLE VI CUMULATIVE GROWTH INCREASE OF CO^ENRICHED DOUGLAS FIR SEEDLINGS D. EFFECT OF TEMPERATURE (0.03% C0 2 only) Irradiance = 7.2 mw cm" PRE- TREATMENT MEAN LENGTH OF NEW GROWTH (cm ) Thermoperiods After 6 weeks pre-treatment After 4 weeks post-treatment TOTAL 25/20 C ' /5 C 0.52' 0.02' 0.54' 5/5 C 0.43' 0.06' 0.49' The data points shown in the table are the means of 55 plants. Analysis of Variance was used for determining statistical differences. Values bearing the same letter within the same column are not significantly different from each other at p =0.01. Plants were pre-treated for six weeks under 8-hour days and four thermoperiods, as shown above. After pre-treatment plants were transferred to a four week post-treatment of 8-hour days (25 C/5 C). All treatments were in normal air (0.03% CO2).

40 25 of experiments 1 and 2 was that it took much longer for plants in experiment 2 to show growth benefits from CC^ enrichment. The delayed response may have been due to the low irradiance under which the trees in experiment 2 were grown (3.4 mw cm-2). If so, it appears that limiting light availability to enriched plants may also limit their ability.to benefit from CO^ enrichment. Plants in experiment 2 did eventually respond to CO^ enrichment, but it took much longer for effects of treatment to become evident. Six-month-old Douglas fir responded to the effects of daylength and CO^ similarly to 1-year-old Douglas fir. Photoperiod had some effect on growth, since the mean heights of long-day seedlings were greater than the mean heights of short-day seedlings (Figure 3). The most notable increases however, were attributabl'e to levels of CO,, enrichment. Under both short and long days, seedlings given intermediate CO,, levels were the tallest, while those given normal CO,, levels were the next tallest. The shortest seedlings were those grown under high C0 2 levels (1.0% and 5.0% C0 2 >. There were no statistical differences between the means of plants grown under 1.0% and 5.0% CO,,, which probably indicates that the maximum effective degree of inhibition lies somewhere between these two levels and that greater inhibition of elongation by higher C0 2 concentrations is unlikely. B. WEIGHT 1. One-year-old trees (a) Effects of 8- and 16-hour daylengths and two CO,, levels on weight An increase in daylength resulted in greater shoot dry weight

41 26 under both low and high CO^ levels (Table VII). However, the highest weights were observed under long days and high CO^, and conversely the lowest weights were observed under short days and low CO^. The significant differences in shoot weight under long days and high CC^ were primarily a result of increases in leaf weight. Stem weight also increased under long days and high CO^t but it could not be shown to be significantly different from the other three treatments. The highest mean values for root dry weight were also observed under long days and high CO^, but there were no significant differences between the four conditions. Additionally, data for trees grown under the above conditions were analyzed using the mathematical relationship known as the shoot: root ratio (Table VIII). The shoot:root ratio has long been used for analyzing whole plant response to various treatments. This ratio reduces two measurements (shoot weight and root weight) to a single measurement which indicates the relative amounts of biomass distributed between the upper and lower portions of the plant. In Table VIII the lowest ratios were those observed for plants maintained under short days and low CC^ levels while the highest ratios were those observed for plants under long days and high CO^ levels. that greater biomass accumulated in the shoot High ratios indicate relative to the root. However, as a comparison of the data in Tables I and VIII shows high shoot:root ratios do not necessarily indicate that increased internodal elongation has also occurred. Although trees which were grown under long days and high CC^ had the highest shoof.root ratios, they had the

42 TABLE VII DRY WEIGHT MEAN VALUES DOUGLAS FIR TREES (1-0 Stock) Grown for 30 days under designated daylength/cx^ combinations -2 Irradiance = 3.4mW cm TREATMENT SHOOT Daylength(h) C02conc. TOTAL (g ) Leaf (mg) Stem (mg) ROOT TOTAL (mg) c C C 407.8' ' 268.4' 361.3' c 603.4' ' l c C The data points shown in the table are the means of 8 plants. Analysis of Variance was used for determining statistical differences. Values bearing the same letter within the same column are not significantly different at p = 0.01.

43 28 TABLE VIII SHOOT : ROOT RATIO (Calculated on grams dry weight) DOUGLAS FIR TREES (1-0 Stock) Grown for 30 days under designated daylength/c^ combinations Irradiance _2 = 3.4 mw cm Daylength (h) CO cone. (%) Shoot/Root at 30 days a b Initial shoot/root = 1.36 for all treatments The data points shown in the table are the means of 8 plants. Analysis of Variance was used for determining statistical differe Values bearing the same letter are not significantly different at p = 0.01.