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The practical application of two dormancy induction trials on douglas-fir and western hemlock container… Wickman, Marise 1985

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THE PRACTICAL APPLICATION OF TWO DORMANCY INDUCTION TRIALS ON DOUGLAS-FUR AND WESTERN HEMLOCK CONTAINER SEEDLINGS by Marise Wickman B .S .F . , University of Brit ish Columbia, 1979 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF FORESTRY l n THE FACULTY OF GRADUATE STUDIES (Faculty of Forestry) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1985 © Marise Wickman, 1985 In p r e s e n t i n g this thesis in partial fu l f i lment of the r e q u i r e m e n t s for an a d v a n c e d d e g r e e at the Univers i ty of Brit ish C o l u m b i a , I agree that the Library shall m a k e it freely avai lable fo r re ference a n d s tudy . I further agree that p e r m i s s i o n for ex tens ive c o p y i n g of this thesis for scholar ly p u r p o s e s may be g ranted by the h e a d of m y d e p a r t m e n t o r by his o r her representat ives . It is u n d e r s t o o d that c o p y i n g o r pub l i ca t ion of this thesis for f inancia l gain shall not b e a l l o w e d w i t h o u t m y wr i t ten p e r m i s s i o n . D e p a r t m e n t of / g / ' g s ^ 6 C / 1 e The Un ivers i ty of Brit ish C o l u m b i a 1956 M a i n M a l l V a n c o u v e r , C a n a d a V 6 T 1Y3 DE-6(3/81) - i i -ABSTRACT Two dormancy induction t r i a l s were conducted in a private container nursery in Saanichton, Bri t i sh Columbia. The f i r s t study examined the effects of photoperiod induced dormancy on morphology, root growth and f ie ld performance of fa l l planted western hemlock (Tsuga heterophyl_1ji (Raf.)Sarg.) and Douglas-fir (Pseudotsuga menzi_esv[ (Mirb.) Franco) seedlings. Various periods of eight hour days, ranging from two to eight weeks, were applied throughout July and August 1983. Outplanting was done in late September. Survival and growth were assessed one year later . The second project investigated the effectiveness of short days, varying levels of moisture stress and a combination of both as dormancy induction techniques for Douglas-fir seedlings. The short day treatment was four weeks of eight hour days. Four levels of predawn moisture stress were: -5, -10, -15 and -25 bars. These classes respectively corresponded to control , l i gh t , medium and severe moisture stress levels . Short days and moisture stress were also combined whereby the four week period of short days followed the moisture stress treatments. These induction treatments were applied in July and August 1984. All seedlings were l i f ted in January 1985 and placed into cold storage for five weeks until March 1985. Morphology, root growth capacity, frost hardiness and dormancy intensity were assessed in January. Root growth capacity and dormancy intensity were again measured in March. In Study I, short days quickly init iated homogeneous budset in both species in approximately three weeks. The average height increment after treatment in i t ia t ion was 3.7 cm in Douglas-fir and 4.2 cm in western hemlock. Short days reduced shoot dry weight and height. Caliper and root dry weight were unaffected. In September a surge in root growth occurred in hemlock seedlings treated with six or eight weeks of short days. The importance of early budset to allow increased root growth prior to a fa l l l i f t was demonstrated. Root growth capacity was similar among al l treatments for both species. The planting survival of western hemlock seedlings increased with increasing weeks of short days. Control plants had 76% survival while the eight week regime had 91%. Survival was similar for a l l treated Douglas-fir seedlings. It ranged from 89% in the two week interval to 98% in the four week regime. One year height increment was s ignif icantly greater in the six and eight week short day treatments for both species. For hemlock, i t ranged from 6.1 cm in the control plants to 10.4 cm in the six week trees. Douglas-fir height increment ranged from 6.4 cm for the control interval to 8.6 cm in the eight week regime. The six and eight week photoregimes produced the best quality hemlock seedlings for this study. Four weeks of short days appeared adequate for Douglas-fir. In Study II short days effectively init iated and maintained budset in Douglas-fir seedlings in four weeks. After six weeks from treatment i n i t i a t i o n , a l ight to severe moisture stress was as effective in controlling height growth. A natural photoperiod with no moisture stress was least effective. - i v-In a comparison of a l l treatment combinations, only the control plants under a natural photoperiod were significantly larger in a l l morphological properties. Short days, moisture stress or a combination of both had similar effects on reducing height, cal iper, shoot dry weight and root dry weight. Unstressed seedlings in a natural daylength had the highest value of root growth capacity. All other treatment combinations had s ignif icantly lower root growth capacity. Only the severe stress under a natural photoperiod s ignif icantly reduced root growth capacity compared to any other treatment. Short days accelerated bud burst in the January and March dormancy intensity tests. Frost hardiness was similar among a l l treatments. Selection of a regime which controlled height growth while maintaining seedling quality was not clearcut. A short photoperiod with no moisture stress was most effective in in i t ia t ing budset. However, few morphological and physiological differences were evident between short day plants and l ight and medium stressed seedlings. -v~ TABLE Or' CONTENTS Thesis abstract i i Table of Contents v List of Tables v i i i List of Figures xi Acknowledgements xiv CHAPTER ONE Introduction 1 1.1 Thesis Objectives 7 1.2 What is Stock Quality? 8 1.2.1 Seedling Morphology 10 1.2.2 Root Growth Capacity 14 1.2.3 Frost Hardiness 20 CHAPTER TWO 2.1 Classical Dormancy 22 2.2 The Complex Interaction Between Hormone Regulators and Physiological Dormancy 24 2.3 The Environmental Role in Dormancy 27 2.3.1 The Phytochrome System 29 2.3.2 Environmental Signals in Dormancy Development . . . . 31 2.3.3 Environmental Factors in Dormancy Release 39 3.0 The Effect of Lif t ing and Storage on Seedling Survival and Growth Performance 44 4.0 Conclusions 49 -vi -CHAPTER THREE The Effect of Photoperiod Induced Dormancy on Morphology, Root Growth and Outplanting Performance of Western Hemlock and Douglas-fir Containerized Seedlings 51 3.1 Introduction 51 3.2 Study Area 53 3.3 Materials and Methods 53 3.3.1 Seedlings 53 3.3.2 The Blackout System 54 3.3.3 Treatments 54 3.3.4 Measurements 55 3.3.5 Stat is t ical Analysis 56 3.4 Nursery Trial Results 56 3.4.1 Greenhouse Climate 56 3.4.2 Rate of Bud Formation 56 3.4.3 Morphology 58 3.4.4 Root Growth Capacity 77 3.4.5 Frost Hardiness 77 3.5 Planting Trial Results 77 3.5.1 Climate 77 3.5.2 Visual Observations 79 3.5.3 Survival and Growth Performance 81 3.6 Discussion 93 3.6.1 Nursery Trial 93 3.6.2 Planting Trial 96 3.7 Conclusions 104 -vi i -CHAPTER FOUR Dormancy Induction of Douglas-fir Containerized Seedlings: A Comparison Between Moisture Stress, Short Days and a Combination of Moisture Stress Followed by Short Days 108 4.1 Introduction 108 4.2 Methods 109 4.2.2 Measurements I l l 4.2.3 Stat ist ical Analysis I l l 4.3 Results 112 4.3.1 Treatments 112 4.3.2 Daily Climate 116 4.3.3 Bud Formation and Incidence of Reflushing 116 4.3.4 Morphology 122 4.3.5 Root Growth Capacity (RGC) 132 4.3.6 Dormancy Intensity 135 4.3.7 Frost Hardiness . . . 139 4.4 Discussion 139 4.5 Conclusions 149 REFERENCES 153 APPENDICES 162 1 162 II 166 I l ia 168 I l ib 170 IV 172 V 174 VI 196 VII 197 v i i i -LIST OF TABLES Table Page No. Table 1.1 1984 proposed stock specifications for a variety of site types 12 Table 1.2 Ministry of Forests stock specifications for 1983 container crops 13 Table 1.3 Index of root growth capacity 19 Table 3.1 The average height growth in Douglas-fir and western hemlock seedlings after short day dormancy commenced until buds formed 61 Table 3.2 Morphology measurements for western hemlock seedlings upon the completion of a l l short day dormancy induction regimes on 17 August 1983 63 Table 3.3 Morphology measurements for Douglas-fir seedlings upon the completion of a l l short day dormancy induction regimes on August 17, 1983 63 Table 3.4 Morphology measurements for western hemlock seedlings four weeks after the completion of a l l photoregime treatments on 19 September 1983 64 Table 3.5 Morphology measurement for Douglas-fir seedlings four weeks after the completion of a l l photoregime treatments on 19 September 1983 64 Table 3.6 Caliper growth in Douglas-fir seedlings after four weeks of conditioning either inside or outside the greenhouse 65 Table 3.7 Average shoot dry weight determinations in western hemlock seedlings which received four weeks of conditioning inside or outside the greenhouse for four weeks after dormancy induction treatments were completed 71 Table 3.8 Average shoot dry weight determinations in Douglas-fir seedlings which received four weeks of conditioning inside or outside the greenhouse for four weeks after dormancy induction treatments were completed 71 Table 3.9 The proportion of sampled western hemlock seedlings where root dry weights conformed to the MOF cull and target standards on 19 September 1983 74 ix Table 3.10 The proportion of sampled Douglas-fir seedlings where root dry weights conformed to the MOF cull and target standards on 17 September 1983 74 Table 3.11 Root growth capacity of Douglas-fir and western hemlock seedlings upon the completion of short day dormancy induction treatments 75 Table 3.12 Daily maximum and minimum temperatures and rainfal l from the last day of planting on 26 September 1983 until 31 October 1983 78 Table 3.13 The incidence of top k i l l , dead or missing terminal buds and needle loss in outplanted western hemlock seedlings. Visual damage was observed in the spring following the f a l l planting 80 Table 3.14 The incidence of top k i l l , dead or missing terminal buds and needle loss in outplanted Douglas-fir seedlings. Visual damage was observed in the spring following the fa l l planting 80 Table 3.15 Survival results for western hemlock one year after planting 86 Table 3.16 Survival results for Douglas-fir one year after planting 86 Table 3.17 Morphology measurements in outplanted western hemlock. Seedlings were assessed after one growing season in October 1984 88 Table 3.18 Morphology measurements in outplanted Douglas-fir. Seedlings were assessed after one growing season in October 1984 88 Table 4.1 Plant moisture stress, styroblock weight, waterloss on a weight basis, and soil water content at the time each treatment was watered 116 Table 4.2 Terminal bud formation in Douglas-fir seedlings maintained under five dormancy induction regimes. Assessment made sixteen days after treatment 121 -X-Table 4.3 The effect of photoperiod and moisture stress on terminal bud formation of Douglas-fir seedlings. Assessment made four weeks after project in i t i a t i on . . . . 121 Table 4.4 Bud formation and incidence of reflushing in eight dormancy induction treatments applied to Douglas-fir seedlings 122 Table 4.5 Morphology measurements of Douglas-fir seedlings at the time of the January 1985 l i f t 126 Table 4.6 The effects of photoperiod and moisture regime on total height in Douglas-fir seedlings 130 Table 4.7 The effect of photoperiod and moisture regime on caliper growth of Douglas-fir seedlings 13 0 Table 4.8 The effect of photoperiod and moisture regime on shoot dry weight accumulations of Douglas-fir seedlings 134 Table 4.9 The effects of photoperiod and moisture regime on root dry weight of Douglas-fir seedlings 134 Table 4.10 The effect of moisture stress on root growth capacity of Douglas-fir seedlings under two photoperiods 136 Table 4.11 The effect of photoperiod and moisture stress on dormancy intensity of Douglas-fir seedlings. Tests were conducted during the January l i f t and after five weeks of cold storage 139 -xi -LIST OF FIGURES Figure 1.1 Periodicity of root growth potential , root growth and shoot growth as i t relates to bud dormancy 15 Figure 1.2 Seasonal changes of root growth potential (RGP), cold hardiness (LT 5 0 ) and water potential at zero turgor (C|Jz) in Douglas-fir seedlings 16 Figure 2.1 Schematic model of hormonal interaction and the the regulation of shoot growth 28 Figure 2.2 The interrelationship between DBB, DRI and ch i l l ing sum. The slope and positioning of the curves will vary with temperature and photoperiod 41 Figure 3.1 The effect of variable weeks of short days (SD) on height growth of Douglas-fir seedlings 59 Figure 3.2 The effect of variable weeks of short days (SD) on height growth of western hemlock seedlings 60 Figure 3.3 The effect of variable weeks of short days (SDO on shoot dry weight of western hemlock seedlings 66 Figure 3.4 The effect of variable weeks of short days (SD) on root growth of western hemlock seedlings 67 Figure 3.5 The effect of variable weeks of short days (SD) on shoot dry weight of Douglas-fir seedlings 68 Figure 3.6 The effect of variable weeks of short days (SD) on root growth of Douglas-fir seedlings 69 Figure 3.7 The effects of variable weeks of short days and conditioning on shoot dry weight of Douglas-fir seedlings in late September 72 Figure 3.8 The September l i f t root dry weights of hemlock seedlings treated with variable weeks of short days. . . . 76 Figure 3.9 One year survival in outplanted western hemlock seedlings treated with variable weeks of short days. . . . 82 Figure 3.10 One year survival of Douglas-fir seedlings treated with variable weeks of short days 83 -xi i -Figure 3.11 Total height, after one year, of outplanted western hemlock seedlings treated with variable weeks of short days 84 Figure 3.12 Total height, after one year, of outplanted Douglas-fir seedlings treated with variable weeks of short days. 85 Figure 3.13 Height increment after one growing season in western hemlock seedlings treated with variable weeks of short days 89 Figure 3.14 Height increment after one growing season in Douglas-fir seedlings treated with variable weeks of short days 9 0 Figure 3.15 Relative height growth (yr - 1 ) after one growing season in western hemlock seedlings treated with variable weeks of short days 91 Figure 3.15 Relative height growth (yr - 1 ) of outplanted Douglas-fir seedlings treated with variable weeks of short days 92 Figure 4.1 The relationship between shoot water potential and soi l water content {%) 117 Figure 4.2 The relationship between water potential and styroblock weight 118 Figure 4.3 Budset incidence in Douglas-fir seedlings after four weeks of treatment with moisture stress, short days or a combination of both 123 Figure 4.4 Incidence of flushed terminal buds in Douglas-fir seedlings after four weeks of treatment with moisture stress and short days 124 Figure 4.5 The final height of Douglas-fir seedlings treated with moisture stress, short days or a combination of both 128 Figure 4.6 The effects of moisture stress and photoperiod on the final caliper measurement of Douglas-fir seedlings in January 1985 129 - x i i i -Figure 4.7 The effects of moisture stress and photoperiod on the final measurement of shoot dry weight of Douglas-fir seedlings in January 1985 132 Figure 4.8 The effects of moisture stress and photoperiod on the final root dry weight measurement of Douglas-fir seedlings in January 1985 133 Figure 4.9 The effects of moisture stress and photoperiod on root growth capacity of Douglas-fir seedlings measured in late January 1985 137 Figure 4.10 The effects of moisture stress and photoperiod pretreatment on dormancy intensity of Douglas-fir seedlings in January 1985 140 Figure 4.11 The effects of moisture stress and photoperiod pretreatment on dormancy intensity of Douglas-fir seedlings in March 1985 141 -xi v-ACKNOWLEDGEMENTS I would l ike to offer a very special thanks to Lynn Husted for her constant moral support and exhuberant assistance in the f ie ld and in the laboratory. I am also grateful to her for in i t ia t ing this graduate research project with CIP Forest Products. I would also l ike to thank Mike Wickman for cheerfully measuring and weighing l i t e r a l l y thousands of forest seedlings; Grace Briggs for processing the data and typing the manuscript; Cathy Haskin for her assistance in the laboratory; and the technical staff of CIP Forest Products for growing and planting the seedlings and conducting f i e ld assessments. I would also l ike to express gratitude to CIP Forest Products for financing the research project and for supplying the technical assistance. Special thanks to Ed McDonald of MacMillan Bloedel Ltd. for donating a l l the hemlock seedlings. F inal ly , I am grateful for the financial support provided to me by the National Science and Engineering Research Council of Canada. 1 CHAPTER ONE INTRODUCTION Reforestation is the most extensively practised s i lv i cu l tura l act ivi ty in Brit i sh Columbia. In spite of a depressed forest economy, the provincial mandate for reforestation remains strong in the face of reductions in the provincial s i lv icul ture budget. In the fiscal year of April 1, 1984 to March 31, 1985 over 100 mil l ion trees were planted. Although i t is doubtful that the present reforestation programme meets a l l industrial and crown requirements, the programme has not been seriously curtai led. Thus, continued government funding demonstrates the pol i t ica l and, to a lesser extent, s i lv icu l tura l importance of reforestation in Brit i sh Columbia. Brit i sh Columbia's reforestation programme has undergone a major change in emphasis since its inception approximately four decades ago. Prior to the "seventies", the planting prescription for Brit ish Columbia's coastal forest regions was: broadcast burn and plant bareroot Douglas-fir ( ^ ^ e u d o t s u £ a _ m e f 2 2 i ^ s ( M i r b . ) Franco). Productivity was the theme. With easy swings of the mattock., a planter generally drove over 1000 bare root seedlings per day into rocky so i l s . Planting quality surveys were quickly done with one gentle pull on the terminal shoot of the seedling. Planting survival was surveyed on thousands of hectares by a brief examination of 25 or so staked seedlings in a plantation. However, the seventies brought in new philosophies towards reforestation. Species prescriptions on a site specific basis slowly evolved with the increasing awareness of ecosystem class i f icat ion and habitat typing. Emphasis on planting productivity partly 2 shifted to include planting quality. More attention was focussed also on stock handling in the f i e l d . Planting checks and survival surveys became more intensive. Along with this shift in emphasis to planting quality, an awareness of the stock's physiological quality increased throughout the late seventies. Foresters demanded better, high quality seedlings. However, in spite of numerous conferences on stock quality, no agreement yet exists on what morphological and physiological attributes comprise a singular, high quality tree. After the 1980 IUFRO Symposium on "Techniques for Evaluating Planting Stock Quality", quality was defined as "fitness for purpose" of ensuring plantation establishment and growth performance (Ritchie 1984a). However, this rather esoteric definition does l i t t l e to create standards to which nursery managers should grow their forest seedlings. A high quality seedling reflects a complexity of physiological and morphological properties. Specifically defining stock quality may prove too idea l i s t ic a task. Stock quality requirements vary with species, s i te , time of planting, environment and numerous other factors. Yet, the search for understanding of stock quality continues. Two symposia recently convened to discuss the seedling attributes which seem correlated with successful plantation establishment and growth performance (Duryea and Landis 1984; Duryea 1985). These attributes are divided into two groups: material attributes and performance attributes (Duryea 1985; Ritchie 1984a). Material attributes are directly measured components which ultimately contribute to seedling performance. Foliar nutrient concentrations, carbohydrate levels and shoot height are only a few 3 examples of these components. Performance attributes reflect the performance of a whole seedling that is subjected to a particular test such as root growth capacity or seedling vigour. At Oregon State University, evaluation tests for both these attributes are either being implemented or are in a process of development. Similar research is being done by the Ministry of Forests but a serious gap continues to exist between research, nurseries and reforestation operations. Part of the problem results from lack of feedback from planting operations to the nurseries. It is in the f ie ld foresters ' , s i l v i c u l t u r i s t s ' and nursery managers' best interests to be aware of the research about the physiological and morphological attributes of forest seedling quality. The track record for planting survival and plantation growth must be improved. Although stock handling and planting quality have probably improved survival , plantation failures and poor growth performance s t i l l present economical and s i lv icu l tura l problems. Although there are numerous factors involved in plantation fai lures : poor site preparation, planting quality, stock handling, s i te quality, summer drought, e tc . ; poor physiological adaptation of the stock to its environment is frequently a common cause (Chavasse 1980; Lavender and Cleary 1974; Nelson and Lavender 1976). In other words, the seedling is physiologically out of synchrony with its environment (Sandvik 1980). An example of this is when stock is early l i f ted and fa i l planted on a high elevation s i te . Poor survival frequently occurs. The seedlings are not suff iciently hardened to withstand the early frosts common in high elevation 4 zones. Physiological vigour or condition of a forest seedling is largely determined by nursery environment and cultural practices. The scheduling of nursery regimes is very important to seedling quality; time and method of dormancy induction, date of l i f t and duration of cold storage can greatly influence seedling physiology and subsequent f ie ld survival and growth performance (Chavasse 1980; Duryea and Lavender 1982; Hermann 1967; Hermann et a l . 1972; Lavender and Cleary 1974; Lavender and Wareing 1972; Lavender and Hermann 1970; Ritchie 1984b, 1982; Timmis 1974; van den Driessche 1983, 1976a, 1969b). Dormant trees are better able to withstand exposure of roots during l i f t i n g , extended periods of cold storage and the harsh environment of a planting s i te . Dormancy is not a steady state; i t is constantly developing or slowly releasing (Campbell 1978). Hence the l i f t i n g and planting window is narrower than the interval between budset and budflush. In addition, dormancy is interrelated with two other important performance attributes: root growth capacity and frost hardiness. Cold hardiness or acclimation is necessary for seedlings to withstand cold storage, outdoor overwintering or the early frosts of fa l l planting. High root growth capacity, the potential ab i l i ty of seedlings to grow new roots, may be necessary to ensure freshly planted seedlings quickly establish new roots prior to the onset of summer drought. The interaction between l i f t i n g date, duration of storage and state of dormancy can greatly affect the level of root growth capacity and degree of frost hardiness (Ritchie 1984a, 1984b, 1985; Ritchie and Dunlap 1980). All of these factors, in turn, can determine survival and outplanting performance. 5 Although the importance of these physiological attributes to the production of high quality seedlings is undeniable, the prime objective of operational forest nurseries remains dogmatically singular: to produce seedlings which meet predetermined morphological size spec i f i ca t ions . Nurseries grade their seedlings on morphological c r i ter ia such as height, caliper and seedling dry weights. Nursery culturing techniques such as fer t i l i za t ion and irr igat ion regimes are commonly prescribed to manipulate these specifications in a forest crop. Height and seedling balance are the main parameters used to gauge seedling growth. Once seedling shoots approach target specifications, height growth is controlled in container and bareroot crops by a r t i f i c a l l y inducing dormancy. This can be done in several ways: drought stress, reduction of nitrogen supply or the application of short days. Moisture stress is probably the most common technique employed in container nurseries. It quickly inhibits the growth processes of cel l elongation and cell d iv i s ion . The reduced water content in the planting medium also reduces nutrient ava i lab i l i ty and plant nutrient uptake. The advantages of moisture stress include ease of implementation and low cost. Possible disadvantages are physiological damage or death i f the stress is too severe. Another method of dormancy induction is photoperiodic control . The mode of action through which short days ini t ia te budset is not well understood. It may affect the balance of growth promoting and inhibit ing hormones so that cell elongation in the apical meristem is reduced. When the photoperiod is shortened in greenhouse nurseries, budset is init iated quickly 6 and uniformly. The development of frost hardiness is enhanced in Douglas-fir when several weeks of eight hour days are applied in the middle of summer. This has important, implications to the cultivation of fa l l scheduled stock. The disadvantages of short day dormancy induction are logist ics and costs. It is also important to minimize l ight leaks in a darkened greenhouse and to prevent high temperatures. Although the purpose of any of these induction techniques is control of height growth, the effect of the regime on physiological quality and outplanting performance must also be considered. There is extensive sc ient i f ic evidence which demonstrates that dormancy is easily ini t ia ted through environmental manipulation; but there are few published studies which examine how specific nursery induction regimes influence seedling morphology, physiology and outplanting performance. This may not be surprising since environmental factors such as l ight intensity, thermoperiod and photoperiod complexly interact to influence dormancy, frost hardiness and various other physiological processes. Because every nursery or greenhouse environment is different, a regime specific to one nursery may produce different results at another nursery. The staff at CIP Forest Products Nursery faced this problem in 1982 where their moisture stress dormancy induction regime produced inconsistent results for controlling height in their Douglas-fir and western hemlock (Tsuga heterophyl1_a (Raf.) Sarg) container stock. They wanted to develop another technique that quickly and homogeneously init iated budset. Consequently, two dormancy induction studies were conducted at the nursery over two consecutive years. The overall intent was to practical ly apply 7 proven physiological principles and documented methods in order to develop an operational nursery regime which effectively controlled height growth through budset in i t ia t ion and also enhanced or at least maintained seedling quality and plantation performance. The f i r s t study was implemented in the summer of 1983 and continued until the fa l l of 1984. A reduced photoperiod of 8 hour days was applied on Douglas-fir and western hemlock seedlings at variable intervals throughout the summer. Morphological characteristics , frost hardiness and root growth capacity were measured at the end of the treatment period. Seedlings were outplanted in the f a l l . Survival and growth performance were assessed during the following autumn in October 1984. Although the application of short days controlled height growth, logist ics and expenses of a "blackout" structure were problems. Consequently in the following summer of 1984, a second study was init iated in order to evaluate the comparative effects of short days and moisture stress. Treatments were again applied throughout the summer. This time, morphology, root growth capacity, frost hardiness and dormancy intensity were assessed just prior to January l i f t and again after five weeks of cold storage. Although CIP Forest Products outplanted these seedlings in May 1985, the planting t r i a l was not included in this graduate research project. 1.1 Thesis Objectives The overall objectives of this thesis are: 1. To develop an operational dormancy induction regime which effectively and homogeneously controlled height growth in Douglas-fir and western hemlock container seedlings. 8 2. To develop a dormancy induction regime which also enhanced or at least maintained seedling quality and outplanting performance. The specific objectives of Study I are: 1. To ensure a "blackout" system, or short days, was operationally effective in in i t i a t ing homogeneous budset. 2. To determine the number of weeks of 8 hour days required to in i t i a te and maintain budset. 3. To determine which treatment interval or short day regime enhanced frost hardiness for fa l l scheduled planting stock. 4. To assess the effects of photoperiod treatment on fal l planting survival and growth. 5. To investigate whether the shortened photoperiod adversely affected root biomass. 6. To evalute the effects of a r t i f i c i a l short days on seedling morphology and root growth capacity. The specific objectives of Study II are: 1. To compare and evaluate the effects of various levels of moisture stress, four weeks of 8 hour days and combinations of both on seedling quality such as: morphology, root growth capacity, frost hardiness and intensity of dormancy. 2. To help develop a moisture stress regime that effectively init iated budset without reducing physiological quality and root morphology. 1.2 What is Stock Quality? CIP Forest Products was interested in short days simply as a tool to stop shoot growth and meet specified height standards for various species and stock types. However, included in this research project were a few basic physiological tests which hopefully reflected more aspects of seedling quality than just morphological characteristics. In this thesis physiological quality was assessed by testing for frost hardiness, root growth capacity and dormancy intensity. Although there are numerous aspects 9 of physiological quality, these tests were selected because they are frequently used in applied research and occasionally in operational nursery production. There is published evidence which demonstrates their usefulness in predicting when to l i f t and store nursery seedlings, and in providing some indication of their fitness for outplanting (Burdett et a l . 1984; Glerum 1985, Duryea 1985; Ritchie 1985; Ritchie and Dunlap 1980). The final test for any seedling crop i s , of course, how well i t survives and performs in the f i e l d . An outplanting t r i a l was conducted for Study I, but time did not permit one for Study II. Consequently, assessment of treatment effectiveness for Study II relies heavily on these physiological tests and on morphological quality. Unfortunately, no definitive test exists to predict how well a crop meets the overall reforestation objectives of surviving and performing well in the f i e l d . There are, however, several which in combination indicate a potential ab i l i ty to grow well when outplanted and thus may reflect a high quality seedling. Whether this potential is actually realized is determined by planting quality, stock handling, time of planting and species and stock type su i tabi l i ty for the site as well as numerous site factors. Therefore, these conditions must be considered when evaluating these predictive tests for fine tuning nursery culture regimes. Because of seasonal climatic var iabi l i ty and operational stock handling, the fine tuning of cultural techniques requires several years of physiological and morphological testing and feedback from the f i e l d . Consequently, the results from these two research studies r ea l i s t i c a l ly will only provide general guidelines to CIP Forest Products Nursery on how to improve dormancy induction techniques of the Douglas-fir and western hemlock container stock. Nursery staff must 10 continue to monitor their induction techniques through quality testing and planting performance t r i a l s in order to develop some flexible methods which effectively in i t ia te budset, control height growth and maintain or enhance physiological quality. There are two proceedings which summarize extensively the numerous aspects and evaluation techniques of stock quality (Duryea 1985; New Zealand Journal of Forestry Science 10(1). In the following pages only those aspects of seedling quality assessed in this research project are briefly reviewed. Dormancy will not be discussed in this section because the next chapter reviews the importance of dormancy to seedling quality. 1.2.1 Seedling Morphology The grading c r i t i e r i a for operational forest nurseries are predominantly based on morphological characteristics (Ritchie 1984a). Height, caliper and root-shoot ratio (R/S) are most commonly used as a basis for cull ing seedlings within any crop. Bud height, root weight and shoot weight are also measured in some nurseries. Although there are extensive research publications which examine the correlation between morphology and outplanting performance, Ritchie (1984a) suggests that comparisons of outplanting performance based upon morphology are largely invalidated because the physiological condition of the seedling is seldom quantified. Definitive conclusions about the relationship between seedling performance and morphology are only valid when a l l test seedlings are in the same physiological state. Nonetheless, i t is generally accepted that i f seedlings 11 are in the same physiological state, large seedlings grow better in the f ie ld but smaller ones survive better (Thompson 1985). Larger trees have a higher photosynthetic capacity for the production of biomass. Smaller trees, however, have lower transpirational demands because of smaller leaf area. Hence, their ab i l i ty to survive f i r s t year summer drought is improved (Hahn and Smith 1983; Thompson 1985). On a site specific basis, the relationship between size, survival and growth performance varies according to brush competition, moisture regime, aspect, soil depth and numerous other environmental factors. For example, on a dry south slope a short compact seedling with lower transpirational demand is desirable but on a north, moist slope, a t a l l seedling is required to compete with the brush. In spite of these site effects, some general trends about seedling morphology are s t i l l evident. Thompson (1985) and Ritchie (1984a) provide reviews on these trends. Height, a measure of photosynthetic capacity and transpirational area, is well correlated with growth performance but a trade off exists between growth and survival (Thompson 1985). Thompson (1985) also reports that a better relationship exists between cal iper, growth performance and survival . Seedling dry weights are correlated in a similar manner as stem diameter. The root-shoot rat io , a measure of seedling balance, is also important. It influences the water balance of a seedling where increasing the rat io , at a given height, improves water uptake to meet transpirational demands (McDonald and Running 1979). In Bri t i sh Columbia, the Ministry of Forests (MOF) has outlined morphological specifications for a number of species stock types for a variety of site types (Table 1.1) Every year the MOF determines 12 Table 1.1 1984 Proposed stock specificiation for a variety of site types. Species Site_Type Stock Type Target Standards Maximum Height - "HttcmT~CaT"Tmm7 TcmJ Fdc Xeric 1+0 PSB 313 17.0 3.2 25.0 Fdc Mesic 2+0 BR 30.0 5.5 40.0 Fdc Brush 2+0 BR 45. 0 6.4 60.0 FDS Alder Rehab. 1+2 BR 60.0 12.0 80.0 Cw Mesic 1+0 PSL 310 20. 0 2.5 27.0 Cw Brush 1+1 PBR 310 50. 0 6. 0 65.0 SS Severe Brush 1+2 BR 60.0 12. 0 80.0 Hw 1+0 PSB 211 17. 0 2.5 25.0 Hw Brush 1+1 PBR 211 35.0 5.0 Ba Species Bg Bn~ Cw~ W Fdc Fdi HaT W Lw"~ W Py s~~ Amabalis Fi r Grand Fi r Noble F i r 2 . 0 West Red 1 3 Cedar 2 . 0 Yellow Cedar Coastal Doug-fir Interior Doug-fir Mountain 11 hemlock 2 . 2 Western 1 4 hemlock. 2 . 2 Western 1 2 . 5 Larch 2 . 0 Lodgepol Pine Yellow Pine Se, SW, S'Frame Se, Sw G'Hse Sitka Spruce Table 1.2. MINISTRY OF FORESTS STOCK SPECIFIC^ Height - Centelmeters (Top Line) Root Collar Diameter - millimeters (Bottom Line) CO r H to m r H r H r H (N to T CO CO co to 10 a. o. a. ""5 1.7 T 6 ~ 2.0 10 2 . 2 T~ 2 . 2 9 1.7 12 2. 12.5 1.8 Cull Standard 2.0 T 3 2.3 12.5 2.4 12.5 2.5 T 2 ~ 2.2 T3~ 2.25 is— TO -2.5 T~ 2.4 10 1.8 15 2.7 14 2.0 TT 2 . 15 3.0 15 3.0 17 3.0 O CO r H O tO <S> CL. O. U 15" 2.2 2.0 CO CO a. T 5 ~ ~ 2.5 15 2.7 T 7 7 5 " 2.5 T 5 -2.5 T 7 T T 2.5 T 7 7 5 " 2.6 T5T3" 3.0 11 2.5 12.5 2.2 15 2.8 15 2.25 Target 10 2 . 2 1775" 3.0 17.5 3.1 17.5 3.2 T B -3.0 S O -3.0 2 T T -3.0 T 5 3 . 2 13 3.0 17 2.5 20 3.0 18 2 . CO in co CL. "TT 2.5 3.5 20 3.7 20 3.5 2 2 3.5 O oo r H O CO o. a. u 2.8 20 2.8 15 2.5 coI F 2 2 2 2 2 T " JT TT 2 2 Maximum Acceptable to r H to co CL. 25 25 25 W W 25 in co co a. w 30" 30 o oo r H O to *t r J O. CO Cu CL. U IONS FOR 1983 CONTAINER CROPS ^ _ Top Dry Weight - grams (Top Line) Root Dry Weight - grams (Bottom Line) Cull Stai idard Tai [get CO i 03 i r H to m O OO r H to in O 00 r H r H r H r H O r H r H r H r H O M to t to <N to to *t CO 03 CO 00 CO co _i a. co co CO CO CL. co CO co co a. CL. a. CL. CL. U CL O, CL. cu u .5 .6 .7 .8 .3 .4 .4 .5 .4 .55 .9 .7 1.0 1.5 .25 .35 .5 .5 .6 .8 .5 .7 1.0 .8 1.0 1.7 .3 .5 .6 .5 .7 .9 .5 .6 .6 .8 .2 .3 .3 .4 .8 1.0 .4 .6 .8 1.0 1.0 1.7 .4 .6 .6 .9 .6 .9 .35 .5 .3 .5 .2 .3 .5 .6 .8 1.0 .3 .4 .5 . 6 .45 .5 .7 .8 .25 .3 .4 .6 .6 .7 .8 .9 .35 .45 .5 .6 .5 .7 .6 .9 .3 .5 .5 .7 .4 .5 .7 .9 .2 .25 .3 .4 .8 .9 1.2 1.0 1.2 1.6 .4 .5 .7 .6 .7 .8 .5 .6 .8 1.0 .2 .25 .4 .5 Maximum Acce] ?tabl( PSB 211 | PSB 313 PSB 41SB PSL 310-CPP 408 1.0 .65 .75 14 morphological standards to which a l l crown land seedlings must be grown (Table 1.2). These specifications are probably altered annually in order to incorporate the previous year's growth curves, any new planting t r i a l results and to probably reflect the rea l i s t ic goals of the current growing season. 1.2.2. Root Growth Capacity High root growth capacity, the potential ab i l i ty of seedlings to grow new roots when placed into a favourable environment, is thought necessary to ensure that freshly planted seedlings quickly establish new roots prior to the onset of summer drought (Ritchie 1985, Ritchie and Dunlap 1980; Sutton 1980). Early exploitation of soil moisture and nutrients may improve a seedling's chance of survival . In studies with white spruce (Picea glauca (Moench) Voss) and lodgepole pine (Pinus_contorta Dougl. ex Loud), a strong correlation between root growth capacity, seedling survival and height growth performance was demonstrated (Burdett, Simpson and Thompson 1983). The periodicity of root growth capacity is related to the stages of dormancy and growth periodicity (Ritchie and Dunlap 1980). Root growth capacity and actual root growth are low throughout the phase of active shoot elongation because of the competition for current photosynthates. Once budset occurs in late summer, current photosynthates are available for root extension as well as for secondary radial growth. Thus, a fa l l surge in root growth commonly occurs throughout September and October for many coniferous trees of the Pacific Northwest. Autumn rainfal l also increases soil moisture supply for root growth. Any successful fa l l planting program should attempt Root Growth Root growth Shoot Growth Dormancy True deepening Dormancy Quies- Shoot Dormancy cence elgation Induction Figure 1.1 Periodicity of root growth potential , root growth shoot growth as i t relates to bud dormancy (Ritchie and Dunlap 1980). 16 1 I I ! L _ _ Nov. Dec. Jan. Feb. Mar. April Figure 1.2 Seasonal changes of root growth potential (RGP), cold hardiness (LT 5 0 ) and water potential at zero turgor (<),2) in Douglas-fir seedlings. Reproduced from Ritchie 1985. 17 to capture this period. Root growth subsequently declines and remains minimal until spring temperatures improve. Root growth commonly peaks prior to spring bud flush. Root growth capacity in Douglas-fir increases during winter as dormant buds accumulate c h i l l i n g hours (Figure 1.1). In coastal Douglas-fir, RGC culminates in January when the ch i l l ing requirement is f u l f i l l e d (Ritchie and Dunlap 1980). Winter l i f t i n g of seedlings has minimum impact on seedlings during this time. One possible explanation is that the high peak of RGC coincides when stress resistance culminates in a seedling (Ritchie 1985). Frost hardiness and drought tolerance are both at their maximum at approximately the same time as RGC (Figure 1.2). Consequently, root growth capacity may also be an indirect measure of seedling tolerance to stress in such species as Douglas-fir (Ritchie 1985). Root growth capacity and stress tolerance rapidly decline with dormancy release. They are at minimum once buds flush. The magnitude and development of root growth capacity varies with species, provenance and stock type (Ritchie 1985). Nursery cultural techniques also affect its development. Time of l i f t i n g in relation to physiological dormancy and to the size of carbohydrate reserves, and the duration of storage affect potential root growth levels (Ritchie 1985; 1982). When seedlings are outplanted numerous factors determine the magnitude of root growth capacity expression (Ritchie 1985; Ritchie and Dunlap 1980). Physiological condition at the time of planting is affected by nursery practices and by subsequent stock handling. Finally the environmental conditions of the planting site wil l influence the extent of new root growth. 18 Soil temperature, moisture and degree of soil compaction a l l influence the expression of root growth potential (Ritchie 1985; Ritchie and Dunlap 1980). In spite of the varying factors which depress root growth in the f i e l d , several studies demonstrate a high correlation between laboratory tested root growth capacity and actual f ie ld survival . Burdett et a l . (1983) reported a correlation coefficient of 0.90 for root growth potential and f i r s t year survival of white spruce and a correlation coefficient of 0.82 for root growth potential and height growth in lodgepole pine. However, Sutton (1983) reported root growth capacity and height growth in outplanted pine and spruce were poorly correlated. Some physiologists question whether high root growth capacity is i t s e l f the direct cause of high survival and growth performance. Lavender (1985, pers. comm.) suggests that root growth capacity may reflect more basic physiological attributes of a seedling and that root growth capacity, i t s e l f , does not account for seedling outplanting response. Richie (1985) suggests that root growth capacity predicts f ie ld performance because i t appears to be correlated with cold hardiness and stress resistance (Figure 1.2), two physiological attributes important to seedling performance. However, no published evidence is presently available which confirms this hypothesis. Methods to test for root growth potential in forest seedlings are reviewed by Ritchie (1984a; 1985). They mostly involve counting the number of new roots and measuring root length on seedlings grown in a controlled environment after a specified time period. The method employed in this research project follows the methodology of Burdett (1979) where seedlings are placed into the controlled environment growth chamber for seven days (Appendix I). The number of new roots are counted and rated into an index 19 TABLE 1.3 Index of root growth capacity (IRG) (Burdett et al . 1983) IRG Description 0 no new roots 1 some new roots, none greater than 1 centimeter (cm) long 2 1-3 new roots over 1 cm long 3 4-10 hew roots over 1 cm long 4 11-30 new roots over 1 cm long 5 31-100 new roots over 1 cm long 6 101-399 new roots over 1 cm long 20 ranging from 0 to 7 (Table 1.3). This quick rating system was designed in order to fac i l i ta te operational nursery testing and is presently employed in many nurseries as routine monitoring. This test was applied in the project as one of three tests to discern treatment differences on physiological quality performance. 1.2.3 Frost Hardiness Frost hardiness refers to a seedling's ab i l i ty to withstand low temperatures. Hardy seedlings are able to tolerate extracelluar ice formation and to avoid lethal intracel lular ice formation (Brown 1980). Intracellular ice usually forms when temperatures rapidly fa l l at a rate greater than 10°C/min and rarely occurs naturally (Weiser 1970). Gradual decreases of 2° or 3°C per hour commonly occur in nature. Ice forms in extracellular spaces where water has the lowest solute concentrations (Weiser 1970). Cell wall permeability increases in hardy tissue to allow the diffusion of water to these extracellular spaces. Hence, the lethal formation of intracel lular ice is avoided. Tree buds also avoid freezing injury by the supercooling of water to temperatures as low as -40°C (Wallner et a l . 1981); although temperatures of - 5 ° are most common (Glerum 1985). Hence supercooling of water is not a major component of frost hardiness development. Cold acclimation occurs in at least two stages (Weiser 1970). In the f i r s t phase, short days and warm temperatures in i t ia te the hardening process through the cessation of shoot growth (Aronsson 1975; Brown 1980; 21 Timmis 1976). Secondly, cold hardiness is enhanced by low temperatures. Although these environmental signals are similar to those which in i t i a te and enhance dormancy, dormancy and hardiness occur independently of one another (Timmis and Worrall 1975). In a third stage, very low temperatures induce extremely hardy tissue. This level of hardiness is quickly lost with warming temperatures (Weiser 1970). Short days may in i t i a te frost hardiness by affecting the level of hormones within leaves (Weiser 1970). Decreasing temperatures possibly affect carbohydrate metabolism, enzymology, protein synthesis as well as plant hormones (Brown 1980). In addition to these environmental s ignals, mineral nutrition and moisture are also implicated as factors in cold acclimation (Aronsson 1980; Benzian 1965; Benzian et a l . 1974; Christersson 1975, 1976, 1978; Timmis 1974). Depending on moisture stress l eve l , moisture stress can enhance, reduce or not affect frost hardiness (Blake et a l . 1978; Glerum 1985, van den Driessche 1969). Potassium may enhance frost hardiness in seedlings through its effect on internal water economy (Christersson 1976, 1975). Drought resistance is another important aspect of frost hardiness. It is frequently necessary for a seedling to be able to tolerate the desiccating effects of frozen s o i l . Seedlings with balanced nutrition appear to acclimate to lower temperatures than seedlings with unbalanced nutrition (Aronsson 1980; Larsen 1978; Timmis 1974). The periodicity of frost hardiness in Douglas-fir follows a similar pattern as that of root growth capacity. The nursery practices of l i f t i n g and cold storage are usually executed when frost hardiness is high because i t is necessary for seedlings to withstand the freezing temperatures of storage or outdoor overwintering. 22 CHAPTER TWO BUD DORMANCY IN FOREST SEEDLINGS 2.1 Classical Dormancy Dormancy is c lass ica l ly defined as "any case in which a tissue predisposed to elongate does not do so (Doorenbos 1953). In forest trees, classical dormancy usually refers to the apical meristem or bud. Three major phases characterize dormancy (Romberger 1963). They include an i n i t i a l stage of quiescence, followed by rest and a subsequent return to quiescence (Lavender and Stafford 1985). In Douglas-fir, budset is init iated in July when terminal buds form in physiological response to summer drought (Blake et a l . 1979; Hanover 1980; Lavender 1981, 1985; Zaerr et a l . 1981). This f i r s t phase of dormancy is called quiescence or imposed dormancy because growth is inhibited exogenously by the environment (Doorenbos 1953; Lavender 1982; Romberger 1963). A return of favourable conditions such as early autumn rain or premature i rr igat ion in the nursery wil l stimulate bud flush and shoot elongation. If this does not happen, several physiological and morphological changes occur (Lavender 1984). The bud continues to develop and grow with the formation of additional leaf primordia (Bachelard 1980; Owens and Molder 1973a, 1973b). Although mitotic act iv i ty is declining in the meristematic apex, cel ls continue to divide in the apex region where the primordia are being formed. Within the tree stem, the cambium also remains active while radial growth and l igni f icat ion of tissue proceeds. 23 Quiescence occurs from mid July until late September in the Pacific Northwest (Lavender 1985). Although moisture stress init iates this phase, the shortening photoperiod of late summer is thought necessary for development into rest (Lavender 1985; Lavender and Stafford 1985). Mild temperatures enhance bud maturation (Cheung 1978; Lavender 1984, 1982; Nelson and Lavender 1979; Sandvik 1980). Coniferous trees of the Pacific Northwest generally enter rest in late September (Lavender 1985). It is imposed by conditions within the bud (Romberger 1963). It develops as the photoperiod shortens while temperatures remain mild. All mitotic act iv i ty ceases within the bud near the end of this stage (Owens and Molder 1973a 1973b; Lavender 1985). Growth inhibitors have greatly accumulated . As temperatures become lower, buds slowly accumulate their ch i l l ing hours which in turn eventually decreases growth inhibitor levels . This eventually releases the bud from rest. Cold resistance begins to develop (Lavender 1985). Buds enter quiescence again in December where the continued accummulation of ch i l l ing hours permits a greater growth response over a wider range of temperatures within the environment (Campbell 1978). The major inhibit ing factor during this phase of quiescence is cold temperature. Chi l l ing fulfil lment and warmer temperatures generally result in active shoot growth around A p r i l . If seedlings have not progressed properly through these stages, buds will not flush with warming spring temperatures. They may burst late in the spring in response to lengthening photoperiod (Campbell 1978; van den Driessche 1976a, 1975). However, competitive advantage to brush invasion can be quickly lost when this occurs in a new plantation. Stress resistance 24 rapidly declines with the cessation of quiescence and the onset of shoot elongation. Nursery disturbance or outplanting is not advisable. The preceding paragraphs were a general review of the physiological stages of dormancy as i t is c lass ica l ly defined. A second definition of dormancy is based on the overall stress resistance of the entire seedling (Lavender 1985). Operationally referred to as hardening off , i t generally develops in Douglas-fir in November and reaches a maximum in January or February. As previously stated, root growth capacity, frost hardiness and winter drought tolerance a l l generally peak in late January in coastal Douglas-fir (Ritchie 1985). The nursery practices of l i f t i n g , storage and planting are recommended during this phase of maximal stress resistance (Lavender 1985; Lavender and Wareing 1972). A third definition is provided by Owens and Molder (1973a) who describe bud dormancy on the basis of mitotic act iv i ty . Buds are considered dormant when cell division ceases within the apex. This occurs approximately from December to April and coincides generally with the development of stress resistance. 2.2 The Complex Interaction Between Hormone Regulators and Physiological Dormancy. In spite of extensive research into the biochemical regulation of plant growth, the exact role of specific plant hormones in dormancy development and release is not clearly understood (Bachelard 1980; Hanover 25 1980; Saunders 1978; Zaerr 1985). Bachelard (1980) suggested that investigations into the hormonal control of bud dormancy have largely focussed on whole bud extracts even though dormancy development and release involve different act iv i t ies within different regions of the apical meristem. He identified a need to examine the biochemistry within specific apical regions in order to further the understanding of hormonal control. Research into the role of plant hormones is also restricted by the d i f f icul ty in extracting and detecting hormones that are present at relatively low concentrations (Zaerr 1985). Saunders (1978) concluded that due to the complexity of hormonal, environmental and genetic interactions, attempts to isolate a specific hormone and assign i t an exact role in dormancy wil l prove frui t less . There are several publications which provide a review of the history and status of plant growth regulator research (Bachelard 1980; Nooden and Weber 1978; Saunders 1978; Wareing and Saunders 1971). It is beyond the scope and objective of this paper to discuss them. Instead, the general concepts of the biochemical control of bud dormancy are presented; although i t must be emphasized that no general hormonal theory is understood or widely accepted (Saunders 1978). The internal regulation of active shoot growth and bud dormancy may be mediated through a balance of growth promoting and growth inhibit ing hormones (Hanover 1980; Lavender and Hermann 197 0). Bud dormancy is imposed when the promoter to inhibitor ratio favours inhibitors (Hanover 1980). High levels of the inhibi tor , abscisic acid (ABA) or "dormin" are frequently but not always correlated with bud dormancy in woody plants (Bachelard 1980; Nooden and Weber 1978). In the buds of Douglas-fir 26 seedlings, Hermann and Lavender (1972) speculated that inhibitor accumulations were very high since exogenous applications of indoleacetic acid (IAA) and gibberellin (GA) were unable to induce bud flush. However, since ABA has not always been detected in dormant buds, Bachelard (1980) suggested that changes in ABA levels are not the only mode of action in bud dormancy. Growth promoters include auxins, gibberellic acids, cytokinins and ethylene. Although auxins influence cell division within the shoot and control correlative inhibit ion of axi l lary buds, their role in terminal bud dormancy is not considered major (Bachelard 1980). Gibberellins (GA) may promote dormancy release where the ratio of GA to ABA determines the in i t ia t ion or release of bud dormancy; low GA to high ABA possibly imposes dormancy while high GA to low ABA may release the shoot apex from growth inhibition (Forycka et a l . 1978). There are at least f i f ty gibberellic acid compounds which exhibit some degree of specifity of species and physiological function (Bachelard 1980). Shoot elongation and flowering are controlled by gibberellins (Zaerr 1985). They are not only metabolized in the shoot but possibly the root as wel l . Cytokinins, which are synthesized in root meristems and in the shoot, are also implicated in dormancy release, and their precise role in dormancy is not understood (Alvim, Hewett and Saunders 1976; Staden and Brown 1978; wareing and Saunders 1971). For example, when root meristems were removed from the roots of Douglas-fir seedlings, l i t t l e effect on bud burst and vigour was evident in seedlings l i f ted and cold stored after October (Hermann and Lavender 1972). A 27 simplistic model of hormone interaction is shown in Figure 2.1, but i t does not indicate how hormone levels interact to regulate specific development act ivi t ies (Bachelard 1980). In spite of this complex interaction between hormones and growth, there have been recent attempts to extract hormones from seedling tissue as a means of assessing seedling vigour and growth potential (Zaerr 1985) and the status of dormancy (Ritchie 1984a). However, the d i f f icul ty in detecting and analyzing low concentrations of hormones has not yet been overcome (Zaerr 1985). 2.3 The Environmental Role in Dormancy In a natural environment the internal regulation of dormancy and growth is finely synchronized with the environment and the seasons. Apical dormancy probably evolved in temperate zone species in order to promote survive cold winter temperatures and summer drought. There are environmental factors which signal to the plant the timely in i t ia t ion of bud dormancy. The following sub-sections are a review of the environment factors which interact with bud dormancy and how the trees "perceive" these st imuli . 28 •> DORMIN t — > old leaves young leaves > GIBBERELLIN •> counteracts effect of gibberellin -> internode extension stem apex > AUXIN — •> mobilisation of nutrients —CYTOKININ root Figure 2.1 Schematic model of hormonal interaction and the regulation of shoot growth. Figure from Bachelard (1980) 29 2.3.1 The Phytochrome System The apical meristem of seedling shoots is a site of perception for environmental signals (Hanover 1980; Hermann and Lavender 1972). Foliage is also a perception site for photoperiodism (Wareing 1956). Light and photoperiod signals are mediated by phytochrome in young seedlings (Hanover 1980; Hillman 1967; Mandoli and Briggs 1984). This pigment is sensitive to spectral l ight quality and the ratio of energy between the red and far red wavelengths (Mandoli and Briggs 1984). Red l ight radiation, prevalent in full sunlight, converts phytochrome into an active form which is sensitive to the absorption of far red l i gh t . Hence this active form is referred to as Pfr. In poor l ight or dark conditions far red l ight dominates. It produces the inactive form (Pr) which is sensitive to the absorption of red l i g h t . Active phytochrome promotes seed germination but suppresses shoot elongation. Response to daylength or photoperiod is a type of phytochrome mediated response. Most of the research on photoperiodism has focussed on horticultural plants such as chrysanthemums. However, since growth periodicity in young seedlings is mediated by phytochrome, an awareness of the importance of l ight quality and quantity on phytochrome will develop understanding on how to regulate seedling growth and dormancy through manipulation of the nursery environment. There are several studies which demonstrate photoperiodic responses of seedlings. Short days induce bud formation in Douglas-fir and western hemlock (Cheung 1973; Hermann et a l . 1972; Lavender and Hermann 197 0; Lavender and Wareing 1972; Lavender and Cleary 1974; Matthews 1977; Nelson 30 and Lavender 1979, 1976; McCreary et a l . 1978; Tanaka 1974, van den Driessche 1969b 1970). When Timmis and Worrall (1975) interrupted these long night treatments with fifteen minutes of red l i ght , bud maturation was delayed in Douglas-fir seedlings. Frost hardiness also declined. However, when far red l ight was radiated immediately after the red l i ght , the red l ight effect was not reversed but accentuated. McCreary et a l . (1978) also reported photoperiodic response in Douglas-fir seedlings. When an eight hour photoperiod was extended with supplemental l ight exceeding 37 |iWcm"2 , the shoot growth of Douglas-fir seedlings increased and bud dormancy prevented. A total photoperiod of eight hours init iated dormancy and enhanced cold hardiness. However, very low l ight leaks decreased hardiness. In western hemlock seedlings, an eight hour photoperiod also inhibited shoot growth and init iated budset, but supplemental l ight as low as l .OiaEm - 2 delayed bud formation (Hauessler 1981). The effect of supplemental far red l ight on growth or dormancy is a c r i t i c a l factor in nurseries especially where northern provenances and species are grown. Extension of the photoperiod with relat ively low l ight intensities (containing far red l ight) promotes shoot extension in northern or high elevation species which require a long photoperiod to maintain shoot growth (Arnott 1979; Tinus 1981). The l ight quality which promotes shoot elongation differs from the l ight quality necessary for photosynthesis (Tinus 1981). Light in the photosynthetic active range occupies the shorter wavelengths from 0.40 to 0.70nm. The energy captured in this range is ut i l ized for photosynthate production which in turn supplies energy for many plant metabolic and biological processes and biomass production. Red l i gh t , at a maximum wave- length of 0.66 \im also occupies this range. However, far 31 red light is beyond this range and is at a maximum of 0.73 0 u i m . It promotes shoot elongation when above a c r i t i c a l l eve l . 2.3.2 Environmental Signals in Dormancy Development The environmental signals which ensure that growth periodicity and dormancy are synchronized with the seasons were briefly discussed during the review on the physiological stages of dormancy. Lavender (1981) provides a concise but extensive review of these environmental factors. Generally, in a natural environment moisture stress or short days inhibit height growth and ini t ia te budset (Hanover 1980, Lavender and Cleary 1974). Continued short days and mild temperatures maintain budset and enhance bud maturation (Sandvik 1980). Short days and cold temperatures promote cold hardiness. Temperatures about 5°C slowly f u l f i l l the ch i l l ing requirement necessary for dormancy release (Campbell 1978, Ritchie 1982). Once this requirement is met, warm temperatures promote bud flush in the spring. The natural sequential changes in photoperiod and thermoperiod that occur from late summer through to spring cause successive, harmonized physiological and anatomical changes within temperate tree species. However, this natural progression of environmental events is frequently altered and delayed in nursery environments, especially in container seedling greenhouses (Lavender and Cleary 1974; Matthews 1977). The moisture regime is tota l ly regulated in greenhouses and partly controlled in bareroot f ie lds . Light quality is reduced in fibreglass greenhouses. Temperatures can vary from the ambient outdoors. Frequently northern latitude or high elevation species are grown in southern coastal nurseries in Brit i sh Columbia. For these seedlings 32 nursery photoperiod does not coincide with seedlot origin and prospective planting site destination. Because the nursery environment may vary from the natural environment, nurserymen should be aware of how their specific nursery environment influences the sequence or progression of physiological dormancy. This fact is especially true when stock is scheduled for early fa l l l i f t ing and outplanting on a high elevation s i te . If dormancy is not induced suff iciently early through environmental manipulation, the seedling grown in a lush, low elevation greenhouse environment will not be physiologically adapted or synchronized with the colder harsh high elevation s i te . In fact, this disharmony between planting environment and the physiological status of the seedling is a major cause of fa l l plantation failures in the Pacific Northwest. The need for synchrony also applies to nurseries which overwinter container stock outside (Glerum 1985) or which place stock into cold storage for the winter (Hermann et a l . 1972). If the seedlings are subjected to cold temperatures prior to entering phyiological dormancy, bud maturation is retarded. If cold hardiness is not suff iciently developed, seedling tissue becomes susceptible to damage from outdoor frosts or cold storage temperatures. Poor survival and/or growth response may result in the following spring. Thus far this section has focussed on the synchrony between seedling physiology and the environment. How does the nurseryman ensure that his seedlings have developed through these phases of dormancy or that his stock is physiologically adapted to its environment whether i t be in a cooler or outplanted in the field? The following paragraphs are a review of research investigating this problem. 33 Scheduling of dormancy induction determines greatly the synchrony between the environment and the physiological stage of dormancy. In the Pacific Northwest dormancy induction should commence in mid June to early July in stock scheduled for fa l l planting (Lavender and Cleary 1974). This will ensure seedlings are suff iciently hardened for an early l i f t and outplanting. For spring scheduled stock, budset must be init iated in sufficient time to allow the buds to mature under the short days and mild temperature conditions of late September and October. The specific date to ini t ia te an induction regime in container greenhouses is largely determined by seedling size where budset is init iated as a means of controlling height growth and the S/R rat io . After reviewing the l iterature i t appears several dormancy induction regimes are available to the nurseryman (Blake et a l . 1979; Cheung 1973; Lavender and Cleary 1974; Matthews 1977; McCreary, Tanaka and Lavender 1978; Nelson and Lavender 1979; Tanaka 1974; Timmus 1974; Timmis and Tanaka 1976; Tinus 1981; Zaerr, Cleary and Jenkinson 1981). Although moisture stress init iates dormancy in Douglas-fir and western hemlock seedlings in natural environments and in bareroot f ie lds , short days are also effective in promoting budset in seedlings grown in environment chambers or greenhouses. In most of the reported studies photoperiods of 8 to 10 hours effectively ini t iated budset in Douglas-fir and western hemlock seedlings. Although a drastic reduction of the photoperiod is recommended (Sandvik 1980), the effects of reducing daily photosynthetic active radiation must be considered. A general decline in root growth was reported in Douglas-fir seedlings receiving short day treatments after October (Lavender and Wareing 1972). The number of active roots, as well as new roots, was reduced. A 34 reduction in photosynthate was suggested as a possible cause. Local empirical t r i a l s have incurred a similar response even when short day treatments were applied in August (Matthews 1977). Early dormancy induction with short day treatments also influences frost hardiness. Compared to a natural summer photoperiod, an 8 hour photoperiod actually enhances cold acclimation in several tree species (McCreary et a l . 1978; Rosvall-Ahnebrink 1981; Tanaka 1972). However, Timmis and Worrall (1975) reported that a six hour photoperiod decreased hardiness in Douglas-fir compared to the eight hour regime. When McCreary et a l . (1978) applied eight weeks of 8 hour days to Douglas-fir, hardiness of the treated seedlings did not differ from that of the control, but the ab i l i ty to quickly acclimate developed shortly after the eight week period. Consequently, a conditioning period following eight weeks of short days was recommended for stock scheduled for early l i f t and fa l l planting. Once short day treatments are applied to actively growing Douglas-f i r and western hemlock seedlings, budset occurs quickly and homogeneously. In a controlled environment of 20°C and eight hour days, western hemlock stopped elongating in three weeks (Cheung 1973). Buds were evident after four weeks. There was 97% bud formation by the seventh week. This response was delayed by temperatures lower or higher than 2 0 ° C . In most nurseries throughout the Pacific Northwest early budset is most commonly ini t iated with moisture stress (Tinus 1981; Zaerr et a l . 1981). Lammas and proleptic growth are minimized compared to regularly watered seedlings. Blake et a l . (1979), however, demonstrated that the level of stress and date of induction affected cold acclimation in Douglas-fir seedlings. A predawn plant moisture stress of -5 to -10 bars 35 enhanced cold hardiness while a level of -10 to -15 bars reduced i t . Cold hardiness was also greater in seedlings where treatments were init iated in mid July compared to treatments started in August and September. Thus an interaction between moisture stress and photoperiod was demonstrated whereby a mild stress applied just prior to a naturally declining photoperiod appeared most effective on hardiness enhancement. Later induction during short days reduced hardiness levels . Van den Driessche (1969b) also reported that moisture stress applied to Douglas-fir seedlings during shortened photoperiods of 8 or 12 hours reduced frost hardiness. Hardiness development was unaffected by stress applied under a long day regime. Seedlings treated with short day regimes exhibited the greatest hardiness. An interaction between moisture stress and container density on frost hardiness of Douglas-fir seedlings also occurs. Timmis and Tanaka (1976) reported that levels were lower in stressed high density seedlings compared to stressed low density seedlings. Unlike Blake et a l . (1979), seedlings which received greater stress acclimated to lower freezing temperatures. Withdrawal of nitrogen (N) f e r t i l i z e r has also proven effective in in i t ia t ing budset although the date of treatment in i t i a t ion strongly interacts with the N withdrawal treatment (Timmis 1974). In a Douglas-fir seedling t r i a l , removal of N in the twelfth week from seedling germination required 45 days for 5 0% of the seedlings to form terminal buds. When the same treatment was applied in the fourteenth week, only 29 days were required for 50% of the sample population to set bud. 36 Timmis (1974) also examined the effects of different balances of NPK f e r t i l i z e r on cold acclimation. Withdrawal of N while maintaining phosphorus and potassium f e r t i l i z e r (PK) drastically reduced frost hardiness to - 1 3 ° C . Nitrogen only and NPK fer t i l i zer s allowed seedlings to obtain hardiness levels of - 2 4 ° C . Interestingly, the seedlings which received no f e r t i l i z e r (-NPK) had a similar hardiness l eve l . However, the greatest hardiness occurred in an application of NPK when K was reduced by 50% of the previous level (NPK 1 / 2 ) . Timmis (1974) concluded that a direct relation- ship existed between cold acclimation and the balance between K and N. Potassium may affect i n i t i a l hardiness levels through its effect on cell sap osmotic potential . However, the N P K 1 / 2 , N and -NPK treatments significantly reduced root growth compared to the standard NPK f e r t i l i z e r . There was also less than 5% bud occurrence in the NPK and NPK 1 / 2 treatments. Timmis (1974) recommended a tissue K/N ratio of 0.6 for hardiness enhancement. At that level the adverse effect on rooth growth would be reduced. Cheung (1973) compared the effectiveness of a l l three major dormancy induction techniques on western hemlock containerized seedlings. Short days induced 100% terminal budset in the shortest time period. For the same time duration, withdrawal of N promoted 51% terminal budset and moisture stress varied between 40% to 60% depending upon time of treatment i n i t i a t i o n . Only the short day treatments consistantly produced dormant seedlings with dark green foliage and higher N content compared to controls. Unfortunately Cheung (1973) only examined morphological attributes. Before any treatment is pronounced better, physiological 37 attributes such as frost hardiness, root growth capacity and growth performance should also be considered. The selection and development of an induction regime must be nursery specific because the alteration of one environmental factor such as photoperiod or moisture regime can interact with other environmental parameters such as irradiance or temperature. A recent study in Sweden best demonstrates the importance of environmental interactions during a dormancy induction regime. Unlike the Pacific Northwest, photoperiod, or short days is an induction technique commonly employed in Sweden. Frost hardiness, seedling s torabi l i ty , survival and growth have signif icantly improved in spruce and pine with the operational application of short days (Aronsson 1975; Rosvall-Ahnebrink 1981; Sandvik 1980). When Norway spruce (Picea abies (L.) Karst.) seedlings were grown in a controlled environment chamber, short days increased fo l iar nitrogen content and accelerated budburst in the following spring (Sandvik 1980). The rate and magnitude of these effects were strongly dependant upon l ight quality and temperature. Day temperatures below an optimum of 2 0°C delayed the induction process. However, low night temperatures during the latter part of the induction phase enhanced cold acclimation especially when irradiance levels were low. Seedling s torabi l i ty was reduced in low radiation levels compared to higher levels . Irradiance during short day treatments also affected fo l iar nitrogen concentrations where nitrogen levels increased with increasing irradiance. Growth potential of the seedlings was positively correlated with fol iar nitrogen. Seedling survival and performance were probably further enhanced by the improved root dry weight which was also associated with high levels of radiation. F inal ly , 38 a provenance interaction was observed. In summary, the effect of short days on seedling physiology and vigour was partly influenced by short day interactions with radiation, temperature regime and provenance. A thermoperiodic effect during dormancy induction was observed in Douglas-fir seedlings. In a nine hour photoperiod, cool day temperatures delayed dormancy induction in coastal provenances while warm day temperatures enhanced i t (Lavender and Overton 1972). In contrast, warm days and coo) nights postponed budset in i t i a t ion in interior provenances. Cool soi l temperature, independent of photoperiod, init iated dormancy in a l l provenances. Lavender and Overton (1972) speculated that cold soil temperatures lowered seedling metabolism and reduced synthesis of cytokinins in the root t ips . The promotor:inhibitor balance shifted to favour growth i nhi b i t ion. The interactions of photoperiod, thermoperiod and moisture regimes were examined in western hemlock (Nelson and Lavender 1976). A combination of eight hour days, moderate moisture stress and warm temperatures (18°C day/ 12°C night) promoted the fastest rate of budset formation. A thermoperiod of 25°C day/20°C night combined with a moderate moisture stress or short days also proved effective in in i t i a t ing rapid budset. The preceding discussion of environmental factors and their inter-action was presented to emphasize the need to develop dormancy induction regimes that are specific to a particular nursery. Thermoperiod, photoperiod l ight quality and moisture regime al l interact to influence the developmental processes of bud dormancy. Dormancy, in turn, is interrelated with root growth capacity and frost hardiness. Thus, nursery practices which affect dormancy may also influence these other two performance attributes. 39 2.3.3. Environmental Factors in Dormancy Release: Chi l l ing Requirement and Bud Burst Chi l l ing requirement is simply the exposure period to Tow temperature that is required to release a seedling from bud dormancy (Nelson and Lavender 1979). It probably evolved as a defense mechanism against buds bursting during a period of warm winter temperatures and subsequent frost k i l l of new growth (Ritchie 1984b). Although the ch i l l ing requirement varies between species and provenances, approximately 2 000 hours of temperatures below 5°C from October to late March satisfy the requirement for most species in the Pacific Northwest (Ritchie 1984b). Under continuous cold conditions of 3 ° C , western hemlock requires eight weeks of c h i l l i n g and Douglas-fir twelve weeks (Lavender 1982; Nelson and Lavender 1979; van den Driessche 1975). Due to fluctuating temperatures in a natural environment this period is longer because warm days may reverse some of the effects of previous ch i l l ing (Nelson and Lavender 1979). Prechil l ing conditions can affect the chi l l ing requirement and subsequent seedling vigour and performance. Pretreatment with short days and mild temperatures prior to ch i l l ing enhanced shoot growth in western hemlock and Douglas-fir seedlings during the following spring (Lavender and Stafford, 1985; Lavender and Wareing 1972; Nelson and Lavender 1979). In addition, when western hemlock seedlings were preconditioned with six weeks of short days, the c h i l l i n g requirement was reduced to four weeks. Pretreatment with long days extended the ch i l l ing requirement from six to eight weeks and inhibited the rate of bud flush. 40 Chi l l ing fulfillment is apparently affected by cold storage. Van den Driessche (1976a) reported that cold storage of Douglas-fir bareroot seedlings did not ful ly satisfy the ch i l l ing requirement. Cold stored seedlings flushed later than naturally chi l led seedlings. However, i f seedlings received 300 hours of natural prechill ing prior to storage, their ab i l i ty to respond to cold storage temperatures was enhanced (Ritchie 1984b; van den Driessche 1975). In summary, there are three major factors which can determine the effect of ch i l l ing on bud dormancy. The time of ch i l l ing and the corresponding physiological state of the seedling are important. As previously stated premature ch i l l ing in the early stages of dormancy can retard bud maturation which in turn reduces shoot growth in the following spring. The temperature of the ch i l l ing and the fluctuation of temperature above 5°C wil l determine when the ch i l l ing requirement is f u l f i l l e d . Campbell (1978) demonstrated that the optimum temperature to satisfy c h i l l i n g requirement actually varies with the accumulation of ch i l l ing hours. After 2 0 days of ch i l l ing at 4 . 4 ° C , 10°C was more effective in reducing the days to bud burst in Douglas-fir. Photoperiod during the storage or ch i l l ing period also affects subsequent bud act iv i ty (Lavender et a l . 1978). Bud act ivity in Douglas-fir increased with exposure to long photoperiod during the ch i l l ing phase. Chi l l ing requirement and bud burst are strongly interrelated. As this relationship is explored, a few terms will be defined. Days to bud burst (DBB) is simply the average number of days for seedlings to break bud (Ritchie 1984a, 1984b). There is a negative logarithmic relationship between DBB and ch i l l ing accumulation (Figure 2.2). In order to l inearize the 41 CHILLING SUM Figure 2.2 The interrelationship between DBB, DRI and c h i l l i n g sum. The slope and positioning of the curves wi l l vary with temperature and photoperiod. Figure from Ritchie 1984a. 42 relationship the reciprocal of DBB is altered into a dormancy release index (DRI) where (Ritchie 1984b): D R I = "DOT The figure 10 represents the number of days for a Douglas-fir seedling in a controlled environment to break bud when the ch i l l ing requirement has been ful ly sat is f ied. The numerator may change with seed source. Thus, a ful ly chi l led seedling wil l exhibit a DRI approaching 1, while a ful ly dormant seedling should have a DRI approaching 0 (Figure 2.2). Thereby, determination of DRI in a growth chamber environment is a means of testing for dormancy intensity and determining whether seedlings have accumulated sufficient ch i l l ing hours. A DRI test , unfortunately, requires time and use of a growth chamber. Because of the interrelationship between ch i l l ing and DRI, recording c h i l l i n g hours may prove a more practical means of estimating dormancy (Ritchie 1984a, 1984b, 1985), although its interpretation can be confounded by fluctuating warm and cold temperatures. As the ch i l l ing requirement becomes f u l f i l l e d , budflush is primarily a temperature mediated response (Worrall and Mergen 1967) where further ch i l l ing incurs a more rapid bud burst over a wider range of temperatures (Campbell 1978; van den Driessche 1975). Variation in bud flush response to temperature can be accounted for by heat sums (Worrall and Mergen 1967). It is the summation of temperature above a c r i t i c a l level such as 0°C multiplied by the duration of the temperature. Once the required quantity of heat sum is achieved within a seedling, budflush commences. Thus the annual spring variation in temperature results in variation in the annual date of budflush. 43 The steepness of the DRI curve shown in Figure 2.2 will change with temperature. Thermoperiod also affects rate of budflush. Van den Driessche (1975) found a daily regime of 18 .5 °C d a y / 7 . 5 ° C night to promote the fastest flushing rate in Douglas-fir seedlings while a regime of 13°C day/13°C night produced the slowest rate. The flushing rate in a warmer daytime temperature of 24°C followed by a colder night temperature of 2°C was in between the f i r s t two thermoperiods. Campbell (1978) suggested that temperature affected buds in two ways: 1. Temperatures between -2°C to +12°C acted as environmental information and promoted minor changes in potential bud development rate. 2. Temperatures greater than 12°C released energy for bud burst and growth. Photoperiod or long days play a minor role in promoting bud release. When long day treatments were applied to Douglas-fir in l ieu of c h i l l i n g , less than 25% of the buds flushed (van den Driessche 1975). Although long days act as a partial and slow substitute for ch i l l ing in trees with part ial ly f u l f i l l e d c h i l l i n g requirements, Campbell (1978) reported that an interaction between photoperiod and ch i l l ing combined to influence budflush at lower spring temperatures. Soil temperature may also play a major role in affecting bud act ivity in Douglas-fir (Lavender et a l . 1973). Seedlings maintained at a soil temperature of 20°C flushed two weeks earl ier than at 5 ° C . Foliar applications of gibberell ic acid accelerated budflush at the colder temperature but not the warmer. The influence of soil temperature on the export of gibberellic substances from the root to the shoot was implicated. 44 To summarize the preceeding section on environmental influence, i t appears that environment, dormancy, ch i l l ing requirement and dormancy release are interrelated. The nurseryman must manipulate the nursery environment to ensure his seedlings develop through the physiological stages of dormancy. The dormancy release index can provide an indication of dormancy intensity. Seedlings should be l i f t ed and stored when dormant, and have received approximately 300 hours of ch i l l ing in the case of Douglas-fir to precondition them to receive further ch i l l ing in cold storage and to enhance storeability and subsequent growth performance in the f i e l d . 3. The Effect of Li f t ing and Storage on Seedling Survival and Growth Performance In the Pacific Northwest seedlings are commonly l i f ted in midwinter and placed into cold storage at temperatures around 0°C for several months (Ritchie 1984b). These nursery practices can greatly affect seedling survival and growth performance in the f ie ld (Burdett and Simpson 1984). The necessity to l i f t during dormancy and high stress resistance creates a f a i r ly narrow l i f t i n g window which varies with provenance, nursery, year and cultural regime (Burdett and Simpson 1984; Ritchie 1982). Burdett and Simpson (1984) suggested that the general recommendation to l i f t during true physiological dormancy was obscure especially when dormancy refers to the physiological state of the bud and not to the whole tree. In addition no quick tests are available to assess the stage of dormancy. Although mitotic indexing provides a relat ively quick indication of dormancy, there are few 45 published reports which correlate mitotic index, a measure of mitotic ac t iv i ty , to date of l i f t i n g and subsequent success in seedling s torabi l i ty and planting survival . Ritchie (1985) recommended that selection of a l i f t i n g date should be based on a measure of stress resistance such as frost tolerance rather than dormancy intensity. Frost hardiness at time of l i f t and the ab i l i ty of seedlings to maintain high RGC during storage are strongly correlated (Burdett and Simpson 1984). Frost hardiness and seedling storabi l i ty are also closely correlated. Operationally, selection of a l i f t i n g date generally corresponds with the period when seedlings have attained a fa ir degree of hardiness. A review of frost hardiness and RGC testing is available in the 1984 Forest Nursery Manual: Production of Bareroot Seedlings (Ritchie 1984a). Several studies have investigated the impact of l i f t i n g date and storage duration on stock quality. Early fa l l l i f t s and storage adversely affect survival and growth because the physical disturbance disrupts the physiological sequence of dormancy development (Lavender 1964). Early l i f t i n g may also prevent seedlings from accumulating sufficient sugars in the fa l l (Krueger and Trappe 1967; Ritchie 1982). Insufficient food reserves reduce the energy necessary for maintenance respiration during storage and subsequent outplanting growth (Krueger and Trappe 1967). Sugars play an important role in the developmental process of cold acclimation. Primarily, they are an energy source for the processes of altering the cell membranes. Increased permeability is necessary to develop frost tolerance. Ritchie (1982) speculated that seedlings l i f ted in the fa l l may have insufficient sugars to gain enough hardiness, but no published studies support this theory. Stress tolerance to l i f t i n g and cold storage is consequently 46 reduced. In one study, early fa l l l i f ted Douglas-fir seedlings exhibited 80% mortality after a few weeks of cold storage in temperatures of 3°C to 5°C (Lavender and Wareing 1972). ' When winter l i f ted stock received the same storage treatment no mortality resulted. Hermann (1967) also observed that the roots of fa l l l i f ted stock were far more sensitive to air exposure during l i f t i n g . Bud burst was delayed with increasing exposure. Cold storage accentuated this effect. In addition, seedlings l i f ted with low RGC are sometimes adversely affected by cold storage . When Douglas-fir seedlings are l i f ted and stored with high RGC, RGC is maintained and even increased far beyond the normal peak period for unstored seedlings. Ritchie (1982) reported that RGC increased for six months in winter l i f ted and stored Douglas-fir seedlings before i t subsequently declined. Long periods of storage are associated with subsequent high seedling mortality and reduced RGC (Ritchie 1982). Reduced seedling performance has been partly attributed to the depletion of carbohydrate reserves to maintain low levels of seedling respiration. Carbohydrate depletion will vary because seedling carbohydrate reserves are influenced by nursery environment, radiation levels , nursery practices such as fe r t i l i za t ion regimes, time of dormancy induction, time of l i f t , etc. Although the level of fo l iar sugars in Douglas-fir was positively correlated with RGC in unstored seedlings, Ritchie (1982) did not find a close correlation between f o l i a r , root and stem carbohydrate (sugar) levels and root growth capacity in stored seedlings. From these results , Ritchie (1982) speculated that RGC reflected the physiological condition within the seedling. He also observed that RGC in Douglas-fir increased with the early 47 stages of dormancy release and decreased during the final stages. It was not shown, however, that this relationship between RGC and dormancy intensity was causal. Ritchie (1982) further speculated that carbohydrate depletion was more related to survival than RGC. When bareroot seedlings are l i f t e d , the roots are disturbed and frequently damaged. However, when root tips were completely removed from bareroot seedlings, l i f t i n g in December and storage had l i t t l e effect upon seedling mortality (Lavender and Wareing 1972). High mortality occurred in the October l i f t . Once again the time of l i f t was a more significant influence on survival . Early l i f t and storage also appeared to accentuate any problems such as root damage, root exposure to air or low RGC. However, the adverse effects of cold dark storage were apparently mediated through the roots as there was l i t t l e reduction in vigour when roots were protected by warm temperature while shoots were stored at 2 ° C . A gradual reduction of the temperature down to 2°C did not reduce seedling mortality. In addition, a daily exposure to low intensities of radiation during storage reduced seedling mortality and improved root growth (Hermann et a l . 1972) and thereby helped to prevent the adverse effects of cold storage. Pretreatment with short days prior to l i f t and storage improved seedling s torabi l i ty and subsequent survival . This may be related to enhanced frost hardiness which improves seedling tolerance of cold temperature. The temperature of the cooler also influences subsequent seedling survival . When Douglas-fir seedlings were stored at varying temperatures from -9°C to 2 ° C , seedlings kept at 2°C exhibited the best survival for coastal and interior provenances (van den Driessche 1976a). Relative shoot growth was also reduced after storage at -2°C compared to 2 ° C . In contrast, 48 Ritchie (1984b) suggested a temperature just below 0°C was better for seedlings stored greater than two months. A subfreezing temperature would reduce seedling maintenance respiration and the rate of carbohydrate depletion. Incidence of storage molds would also decrease. Date of l i f t i n g and cold storage influences the process of dormancy release (Ritchie 1984a; 1984b). In one study on Douglas-fir, Ritchie (1984b) examined how DRI changed over time in naturally chi l led and cooler stored seedlings. As natural ch i l l ing accumulated, DRI progressed towards 1. However, DRI changed more slowly in stored seedlings. The delay was attributed to the temperature of the cooler ( - 1 ° C ) . The optimum temperature for dormancy release ranges from 4°C to 6°C (van den Driessche 1975). Ritchie (1984b) concluded that this delay in dormancy release is desirable when stock is scheduled for late spring planting. Cold storage, therefore, can widen the planting window compared to overwintering of stock in outside beds. By late spring overwintered stock is commonly post dormant, an undesirable physiological state for planting. Ritchie (1984b) also demonstrated that cooler stored, high elevation seedlings were released from dormancy faster than low elevation stock. This is unfortunate since high elevation sites are frequently planted last due to lingering snowpacks (Ritchie 1984b). To conclude, winter l i f t i n g and cold storage practices can improve or at least maintain seedling quality for several months into spring reforestation programmes. RGC can improve and remain high while seedlings are stored at temperatures around 0°C. Release from bud dormancy is also delayed, an advantage in stock scheduled for planting in late spring. Before these advantages of cold storage are realized, the nursery manager must 49 schedule l i f t i n g and storage practices when seedlings are dormant, and when stress resistance and RGC are high. 4. Conclusions The nursery operations of l i f t i n g and storage must be implemented when seedlings are dormant and exhibit high stress resistance. Nursery managers should be aware of how their specific nursery environment affects the development of physiological dormancy. A visual examaination of budset wil l not provide this information because several complex physiological, biochemical and anatomical events continuously occur within the bud from the time of its formation to the time of flushing. Dormancy is also interrelated with root growth capacity, frost hardiness and growth performance. Thus, there is a need to test for the material and performance attributes of nursery seedlings in order to determine the overall effect of nursery regimes on dormancy and general seedling vigour. A number of evaluation tests already exists and several more are in a process of development. Root growth capacity, frost hardiness, drought resistance, dormancy intensity and mineral nutrition are only a few of the tests presently ava i lab le to the nursery manager. Yet, one might ask: Do these tests mean anything? Strong evidence already exists that demonstrates the close correlation between frost hardiness and successful seedling storage; and between high RGC and seedling survival as well as height growth performance. With a knowledge of basic physiological principles , a nurseryman could employ these tests to track the effect of nursery practices and their scheduling on stock quality. However, these actions are meaningless unless feedback is provided from the f i e l d . 50 Although there is T i t t l e agreement among scientis ts , foresters and nurserymen on the definition of stock quality, a l l acknowlege the need to improve seedling quality as a means of increasing planting survival and growth performance. If communication improved between these groups, the f i r s t impediment towards obtaining this objective would be removed. A second blockage is a general lack of monetary commitment towards improving stock quality. Education, development and implementation of evaluation tests, and improved monitoring of new plantations a l l require government and industrial funding. Only once these funds are available, can we say there is a strong s i lv icu l tura l commitment towards the regeneration of Brit ish Columbia's forest land. 51 CHAPTER THREE STUDY ONE THE EFFECT OF PHOTOPERIOD INDUCED DORMANCY ON MORPHOLOGY, ROOT GROWTH AND OUTPLANTING PERFORMANCE OF WESTERN HEMLOCK AND DOUGLAS-FIR CONTAINERIZED SEEDLINGS 3.1 Introduction In greenhouse nurseries i t is possible to regulate the environmental parameters of photoperiod, moisture and temperature to prematurely induce dormancy and enhance frost hardiness (Sandvik 1980). This practice would prepare fa l l scheduled seedlings for the early frosts of high elevation s ites . Moisture stress is already used in many operational nurseries to in i t ia te early budset as a means of regulating height and seedling balance. However, moisture stress has variable effects on frost hardiness. Blake et a l . (1979) found that a mild stress of -5 to -10 bars enhanced cold acclimation in Douglas-fir seedlings compared to regularly watered plants but a more severe stress of -10 to -15 bars reduced hardiness levels to that of control. Van den Driessche (1959b) reported that reduced moisture supply did not hasten hardening off in Douglas-fir. Moisture stress, applied in an 8 or 12 hour photoperiod, actually reduced hardiness compared to frequent i rr igat ion in a short photoperiod. Dormancy was init iated and hardiness enhanced by eight weeks of 8 hour days at warm temperatures followed by a period of cooler temperatures. Several other studies indicate that short day treatments ini t ia te budset and enhance hardiness. Dormling et a l . (1968) demonstrated this effect on Norway spruce. The importance of warm temperatures immediately following budset to improve 52 bud maturation was also observed. Fall planting survival of Norway spruce and Scots pine were successfully improved by treating seedlings with four weeks of 8 hour days in mid July (Rosvall-Ahnebrink 1981). Several studies demonstrated the effectiveness of short days in in i t ia t ing dormancy and enhancing hardiness in Douglas-fir (Lavender and Wareing 1972; McCreary et a l . 1978; Tanaka 1974, van den Driessche 1969b) and in western hemlock (Cheung 1973, 1978; Nelson and Lavender 1976; Haeussler 1981). Interaction between photoperiod and l ight intensity, l ight leakage and temperature have also been examined. McCreary et a l . (1978) and Hauessler (1981) indicated that low intensity l ight leaks during the dark period delay dormancy. Light intensity and temperature also interact with short days to affect the rate of hardening (McCreary et a l . 1978; Sandvik 1980, van den Driessche 197 0). Generally, high irradiance, warm days and cool nights apparently interact with short days to hasten the hardening process while cooler temperatures in the following weeks enhance hardening. In spite of extensive applied research, short day treatments generally have not made a transition into operational nurseries. Although Sweden has implemented short days as an operational cultural too l , only a few nurseries employ the technique in Brit i sh Columbia. Due to inconsistent results with drought stressing, the CIP Forest Products private nursery expressed interest in developing short days as an operational dormancy induction technique for western hemlock and Douglas-fir. However, before any costly investment was made, an operational t r i a l was established in order to develop a short day regime which would successfully in i t ia te budset and maintain or enhance seedling quality, especially frost hardiness and root 53 growth capacity. The influence of short days on survival and outplanting performance of fa l l scheduled stock was also investigated. 3.2 Study Area The nursery t r i a l was conducted at the CIP Forest Products' private nursery in Saanichton, Brit i sh Columbia. It is located approximately 100 metres (m) above sea level on the southern t ip of Vancouver Island. The seedlings were grown and treated in a permanent fibreglass structure greenhouse. Irrigation and fe r t i l i za t ion was done by an overhead boom system. The planting t r i a l was located in the Robertson Valley about thirty miles southwest of Lake Cowichan on Vancouver Island, Brit ish Columbia. Fifteen plots were situated on a southeast aspect at an approximate elevation of 700 m. The study site is in the Montane Variant of the Wetter Coastal Western Hemlock Biogeoclimatic Subzone. A 50X slope, the landform is a colluvial veneer over igneous bedrock. According to the CIP biophysical c lass i f icat ion, site productivity is poor to medium. 3.3 Materials and Methods 3.3.1. Seedlings Western hemlock seed was operationally sown in 211 styroblock containers in late February, 1983 at Koksilah Nursery in Duncan, Br i t i sh Columbia. The seed was from seedlot 2248 collected from seedzone 1010 at an elevation of 122 m. The seedlings were transported to CIP Forest Products' Nursery in the middle of May, 1983. Coastal Douglas-fir seedlot 4371 was sown in 313 styroblock containers on 30 March, 1983 at CIP Forest Products 54 Nursery. The seed came from seedzone 1020 at an elevation of 750 m. All seedlings received standard f e r t i l i z e r and irr igat ion regimes from the time of sowing until the time of l i f t i n g for a fa l l scheduled plant (Appendix III). 3.3.2. The Blackout System A 'blackout' system was necessary to reduce the natural photo-period to a short day of eight hours. A large wooden frame was constructed over a greenhouse bench section. It was covered with black plast ic . To reduce the buildup of high temperature the outside plastic was coated with a white latex paint to reflect the high intensity radiation of the late afternoon. A ventilation system was also installed to exhaust hot a i r . To ensure heat was not building up, temperatures were monitored with a hygrothermograph. The eight hour day started at 7:30 a.m. and finished at 3.30 p.m. Light leaks were monitored with a LIC0R photometer. 3.3.3. Treatments On two separate species of seedlings, five regimes of shortened photoperiod and two post treatment conditionings were factorial ly applied in a completely randomized design. Douglas-fir and western hemlock seedlings received variable weeks of eight hour days. The five regimes were: 0, 2, 4, 5 and 8 weeks of short days. The eight week treatment started on 23 June 1983 and each regime respectively commenced every following two weeks, so that a l l treatments finished on 17 August 1983. Each ^ treatment was applied to 500 Douglas-fir seedlings and 250 western hemlock seedlings. After 17 August 1983, a l l regimes underwent a four week conditioning and hardening off 55 period before the fa l l outplanting. Half of a l l treatments were maintained inside the fibreglass greenhouse and half were outside in ful l sunlight. Seedlings from al l treatments were outplanted in the third week of September. The treatments were then factorial ly arranged in a completely randomized block design. Ten Douglas-fir plots each contained ten seedlings from each treatment while five western hemlock plots each had ten seedlings from al l treatments. A hygrothermograph was maintained on site until snow fa l l accumulated. 3.3.4 Measurements Root and shoot dry weights, height and caliper were measured at the start of each treatment period. Height and caliper were recorded every subsequent week and the dry weights once every two weeks. Ten seedlings per treatment were sampled for each measurement. On 17 August, 1983 ten seedlings from each treatment were tested for root growth capacity and frost hardiness. The procedure for root growth capacity testing is outlined in Appendix I; i t is the standard Ministry of Forests procedure. Sample seedlings were placed into a controlled environment for one week. The number of new roots were counted. Ten trees from al l treatments were also placed into a freezer chest and subjected to a freezing temperature of - 5 ° C . The seedlings were then maintained in the nursery greenhouse for two weeks to be assessed for needle, bud and stem damage. The frost hardiness test was again conducted on 19 September, just prior to the fa l l l i f t . No f a c i l i t i e s were available to assess root growth capacity at that time. For the planting t r i a l , winter damage was assessed in May once the snow melted. Fall survival and growth performance were recorded in October 56 1984. Growth measurements included total height, height increment and cal i per. 3.3.5 Statist ical Analysis Analyses of variance (ANOVA) were conducted on a l l variables. The ANOVA assumptions of homoescasdicity and normal distribution of data were generally met (Appendix IV). ANOVA tables are reported in Appendix V. When the F ratio was significant in an ANOVA, treatment means were compared by Newman-Keul's multiple range test. 3.4 Nursery Trial Results 3.4.1 Greenhouse Climate Daily maximum temperatures in the blackout system ranged from 17°C to 32 °C . The average daily temperature for the eight week photoregime period was 2 6 ° C . Temperatures exceeded 30°C on only two days. Nightly minimum temperatures varied between 11°C and 18°C for an average temperature of 14 °C . These temperatures were similar to those in the greenhouse bench where seedlings were grown under a natural photoperiod (Appendix VII). 3.4.2 Rate of Bud Formation Short days or reduced photoperiod was an effective tool to control height growth in Douglas-fir and western hemlock container stock. In Douglas-fir, soft brown buds formed and became visible in two to three weeks from treatment i n i t i a t i o n . The red, pointed buds characteristic of this species were observed in three to four weeks. Date of treatment in i t i a t ion appeared to influence the rate of budset where treatments init iated in late 57 June or early July produced buds on seedlings in about three weeks and treatments started in Tate July or early August produced buds on seedlings in about two weeks. This most l ike ly reflects the effect of the naturally declining photoperiod on slowly in i t ia t ing budset, especially since 20% of the Douglas-fir control seedlings had terminal buds when the photoregimes were completed on 17 August 1983. On this date, buds had formed on a l l Douglas-fir seedlings in the eight and six week regimes. The four week regime had 95% incidence of budset while the two week short day interval had buds on 80% of the plants. Terminal buds of western hemlock seedlings apparently formed at a slower rate. However, new buds on hemlock are d i f f i cu l t to detect. Short days, regardless of date of treatment i n i t i a t i o n , produced detectable terminal buds in about four weeks. When a l l regimes were completed on 17 August 1983 a l l seedlings in the six and eight week short day intervals exhibited terminal buds while only 55% of the plants in the four week regime had detectable terminal buds. Buds were not evident in the two week regime or control seedlings at that time. Four weeks after the completion of the photoregimes, terminal buds had formed on a l l hemlock seedlings but controls. There was no incidence of a second flush in any of the treatment regimes for either the inside or outside conditioning. In the treated Douglas-fir seedlings, the occurrence of proleptic and lammas growth was less than 1%. Terminal buds were evident in v ir tual ly a l l treated seedlings regardless of inside or outside conditioning. Terminal buds had i n i t i a l l y formed on 90% of a l l control Douglas-fir seedlings by this date, but 25% of these seedlings also underwent a second flush by this time. 58 3.4.3 Morphology Trends in height growth throughout the treatment period until the fa l l plant are presented in Figures 3.1 and 3.2. Douglas-fir height generally levelled off in three weeks when the photoregime was init iated in Tate June or early July. Height growth stopped in about two weeks for the four and two week regimes. These treatments were started in late July and early August. A similar trend was observed for the western hemlock seedlings where the six and eight week treatments init iated in late June or early July, respectively, stopped height growth in three weeks while the four and two week treated plants, treatments started in late July and early August, ceased shoot elongation in about two weeks. Since the overall purpose of operational dormancy induction is control of height growth, i t is important to know how much height growth is expected once treatment is in i t i a ted . This information would help guide a nurseryman when to commence the induction process. The range of height growth after treatment in i t i a t ion is reported in Table 3.1. The average height growth for a l l treated Douglas-fir seedlings was 3.7 cm and for western hemlock 4.2 cm. There was no apparent correlation between date of treatment in i t i a t ion and magnitude of height growth. Due to the staggered arrangement of treatment in i t i a t ion dates total height was s ignif icantly different between most of the treatments for the August and September sample periods whereby early init iated treatments were s ignif icantly shorter than later regimes and the control. All morphological results are found in Tables 3.2, 3.3, 3.4 and 3.5. 59 Figure 3.1. The effect of variable weeks of short days (SD) on height growth of Douglas-fir seedlings. 60 Figure 3.2. The effect of variable weeks of short days (SD) on height growth of western hemlock seedlings. 61 Table 3.1. The average height growth in Douglas-fir and western western hemlock seedlings after short day dormancy commenced until buds formed. PHOTOREGIME AVERAGE HEIGHT GROWTH (cm) (weeks of short days) Douglas-fir Western hemlock Control Two 2.5 4.7 Four 3.9 4.4 Six 5.4 4.1 Eight 3.2 3.7 Average 3.7 4.2 62 Caliper measurements in the Douglas-fir seedlings were unaffected by the photoperiod treatments (Tables 3.3 and 3.5). However, seedlings conditioned outside of the greenhouse had s ta t i s t i ca l ly larger calipers than plants conditioned inside the greenhouse (Table 3.6). This significant difference, an average of 0.21 mm, probably is not operationally s ignif icant. Conditioning or photoperiod did not influence caliper growth in the western hemlock seedlings (Table 3.2 and 3.4). Trends in shoot and root biomass, as measured on a dry weight basis, are demonstrated in Figures 3.3, 3.4, 3.5 and 3.6. Shoot dry weight accumulation generally slowed down and occasionally levelled off in treated western hemlock seedlings until the a r t i f i c i a l photoperiod stopped on 17 August 1983. After this time, shoot dry weight increased in a l l treatments until they were l i f ted in the third week of September. Shoot dry weight accumulation generally slowed down in Douglas-fir plants placed under short day regimes. With the exception of the eight week regime, shoot dry weights continued to increase until the seedlings were l i f ted in September throughout a l l photoregimes including control until the seedlings were l i f ted in September. In both species root dry weight accumulated at similar rates for a l l treatments. However, once the treatments were completed on 17 August, 1983, western hemlock root dry weights greatly increased in the six and eight week photoregimes. Root dry weight also increased but at a slower rate, in a l l other treatments. A surge in root growth also occurred in Douglas-fir after the shortened photoperiod resumed to its natural length. 63 Table 3.2. Morphology measurements for western hemlock seedlings upon the completion of a l l short day dormancy induction regimes on 17 August 1983. MEASUREMENT PHOTOREGIME (weeks) Control Two Four Six Eight Height (cm) 21.3 a 23.O3 17.4b 17.0 b c 14.3C Caliper (mm) 2.55a 2.37 a b 2.15b 2.45 a b 2.38 a b Shoot dry weight (g) 0.84a 0.77 a b 0.53b 0.59 a b 0.5 C5 Root dry weight (g) 0.27a 0.21a 0.2 O3 0.18a 0.22a Values within rows followed by the same letter are not s ignif icantly different at p = 0.05. Table 3.3. Morphology measurements for Douglas-fir seedlings upon the completion of a l l short day dormancy induction regimes on 17 August 1983. MEASUREMENT PHOTOREGIME (weeks) Control Two Four Six Eight Height (cm) 20 .7 a b * 22.2a 18.7 b c 16.5 c d 13.4d Caliper (mm) 2.53a 2.51 a 2.54a 2.76a 2.5 O9 Shoot dry weight (g) 1.06a 1.13a 0.98a 0.829 0.7 3a Root dry weight (g) 0.283 0.3 O3 0.26a 0.293 0.3 4a * Values within rows followed by the same letter are not s ignif icantly different at p = 0.05. 64 Table 3.4. Morphology measurements for western hemlock seedlings four weeks after the completion of al l photoregime treatments on 19 September 1983. MEASUREMENT PHOTOREGIME (weeks) Control Two Four Six Eight Height (cm) 22 .1 a b * 23.9 a 20.0 b c 18.8C 14.9d Caliper (mm) 2.94 a b 3.09a 3.0cPb 2.94 a b 2.59b Shoot dry weight (g) 1.14a 1.20* 1.00 a b 1.07 a b 0.78b Root dry weight (g) 0.43a 0.42a 0.42a 0.6 & 0.52a * Values within rows followed by the same letter are not s ignif icantly di fferent at p = 0. 05. Table 3.5. Morphology measurements for Douglas-fir seedlings four weeks after the completion of a l l photoregime treatments on 19 September 1983. MEASUREMENT PHOTOREGIME (weeks) Control Two Four Six Eight Height (cm) a* 22.9 a 22.4a 17.5b 16.3b 12.9C Caliper (mm) 3.29a 3.44a 3.27a 3.38a 3.07 a Shoot dry weight (g) 1.76 a b 1.8 0* 1.52 a b 1.38b 0.88C Root dry weight (g) 0.61a 0.61a 0.66a 0.7 4a 0.63a * Values within rows followed by the same letter are not s ignif icantly different at p = 0.05. 65 Table 3.6. Caliper growth in Douglas-fir seedlings after four weeks of conditioning either inside or outside the greenhouse. PHOTOREGIME CALIPER GROWTH AFTER FOUR WEEKS OF CONDITIONING (weeks) Inside (mm) Outside (mm) Control Two Four Six Eight Average 3.13 3.48 2.97 3.27 3.06 3.18 a* 3.46 3.40 3.57 3.47 3.08 3.40b * Values followed by same letter are not s ignif icantly different p = 0. 05. 66 Figure 3.3. The effect of variable weeks of short days (SD) on shoot dry weight of western hemlock seedlings. 67 0.8 0.7-0.0 J 1 1 1 1 1 1 r 0 2 4 6 8 10 12 WEEKS FROM TREATMENT INITIATION Figure 3.4. The effect of variable weeks of short days (SD) on root growth of western hemlock seedlings. 68 2-, 2 4 6 8 10 WEEKS FROM TREATMENT INITIATION Figure 3.5. The effect of variable weeks of short days (SD) on shoot dry weight of Douglas-fir seedlings. 69 Figure 3.6. The effect of variable weeks of short days (SD) on root growth of Douglas-fir seedlings. 70 Short days generally reduced shoot dry weight of western hemlock, at both the 17 August and 19 September period (Table 3.2 and 3.4). However, shoot dry weight also varied between some treatments because of the staggered arrangement of treatment in i t i a t ion dates. The eight week regime exhibited s igni f ica l ly reduced shoot dry weight compared to that of control because height growth stopped in this interval several weeks before i t ceased in control seedlings. That i s , the eight week seedlings were s ignif icantly shorter. The two week seedlings were not s igni f ica l ly different in shoot dry weight from control plants because height growth stopped within a similar time period; hence total heights were also the same. The effect of short days or the effect of the early date of treatment in i t i a t ion on shoot dry weight was also evident four weeks after the photoregimes were completed. The eight week regime had signif icantly less shoot dry weight than control seedlings. All other treatments did not differ s ignificantly from controls. The type of conditioning influenced dry weight determinations. Outdoor conditioned western hemlock seedlings had significantly higher shoot dry weights than indoor conditioned plants (Table 3.7). When the photoregimes were completed on 17 August 1983 shoot dry weights in Douglas-fir generally declined with increasing weeks of photoregime (Table 3.3, Figure 3.7). This trend again reflects either the effects of the staggered treatment in i t ia t ion date on height growth or the effect of increasing intervals of short days. The differences were not s ignif icant, however, because of the wide variation about the treatment means. Four weeks after this sample period, there were significant differences in shoot dry weights between treatments (Table 3.5). The eight week regime, init iated in late June, had significantly less shoot biomass 71 Table 3.7. Average shoot dry weights in western hemlock seedlings which received four weeks of conditioning conditioning inside or outside the greenhouse after dormancy induction treatments were completed. PHOTOREGIME (weeks) SHOOT DRY WEIGHT AFTER FOUR WEEKS OF CONDITIONING Inside (g) Outside (g) Control Two Four Six Eight 1.04 1.05 0.85 1.06 0.71 1.24 1.36 1.16 1.08 0.81 Average 0.94 1.13° Values followed by same letter are not s ignif icantly different p = 0.05. Table 3.8. Average shoot dry weights in Douglas-fir seedlings which received four weeks of conditioning inside or outside the greenhouse after dormancy induction treatments were completed. PHOTOREGIME SHOOT DRY WEIGHT AFTER FOUR WEEKS OF CONDITIONING (weeks) Inside (g) Outside (g) Control 1.53 2.00 Two 1.60 2.00 Four 1.26 1.78 Six 1.36 1.39 Eight 0.87 0.89 Average b* 1 . 3 2 ° 1.61a * Values followed by same letter are not s ignif icantly different p = 0.05. 72 2 . 5 -2 . 3 ->-C O 2 .1 -1.9 -t- 1.7-1 o 1.5 -8 1.1-0 . 9 -0 . 7 -Legend A Outs ide C o n d H i o n i n g X Inside C o n d i t i o n i n g 0 . 5 -2 4 6 PHOTOPERIOD REGIME (WKS) 8 Figure 3.7. The effects of variable weeks of short days and conditioning on shoot dry weight of Douglas-fir seedlings in late September. (Vertical bars indicate standard error of the mean. 73 than a l l other later ini t iated photoregimes. The six week regime was also significantly less than the control . Once again, outdoor conditioning significantly enhanced shoot dry weight throughout a l l photoregime treatments (Table 3.8). When a l l photoregimes were just completed, short days had not affected root growth, as measured by root dry weight, of either species (Table 3.2 and 3.3). That i s , for both species there were no significant differences between treatments on 17 August 1983. After four weeks of conditioning, no photoperiod effects on root dry weight were evident for Douglas-fir. However, the influence of short day treatments on western hemlock root dry weight was evident although not s t a t i s t i ca l ly shown by analysis of variance. The six and eight week regimes had greater root masses than the two, four and control treatments (Figure 3.8). To interpret this data for operational purposes, every root dry weight measurement for each treatment was compared to the 1983 MOF western hemlock cull standard of 0.3g and the MOF target standard of 0.5g (Table 3.9). The proportion of seedlings exceeding the root dry weight cull standard for a 211 styroblock container increased with the increasing weeks of short days. Only 65% of the control and two week treated plants met this standard while 90% and 100% of the six and eight week seedlings exceeded this guideline. In addition, a greater proportion of sample seedlings actually conformed to the MOF target standard in the six and eight week photoregimes. The 1983 cull and target standards for Douglas-fir in 311 styroblock containers are 0.4 and 0.6 g. A high proportion of Douglas-fir sample trees exceeded the cull standard throughout a l l treatments 74 Table 3.9. The proportion of sampled western hemlock seedlings where root dry weights conformed to the MOF cull and target standards on 19 September 1983. PHOTOREGIME (weeks) % ABOVE ROOT DRY WEIGHT CULL STANDARD (0.3g) % ABOVE ROOT DRY WEIGHT TARGET STANDARD (0.5g) Control 65 20 Two 65 35 Four 60 35 Six 90 75 Eight 100 55 Table 3.10. The proportion of sampled Douglas-fir seedlings where root dry weights conformed to the MOF cull and target standards on 17 September 1983. PHOTOREGIME % ABOVE ROOT DRY WEIGHT % ABOVE ROOT DRY WEIGHT (weeks) CULL STANDARD (0.4g) TARGET STANDARD (0.6g) Control 90 55 Two 100 5 0 Four 80 60 Six 95 75 Eight 95 70 75 Table 3.11. Root growth capacity of Douglas-fir and western hemlock seedlings upon the completion of short day dormancy induction treatments. ROOT GROWTH CAPACITY PHOTOREGIME (weeks)  MEASUREMENT Control Two Four Six Eight Douglas-fir 4.6 a 3 . 0 ° 3.4 b C 4 .2 a b 4.8a Western hemlock 3.7 a 3.3 a 4.2 a 3.7 a 3.7 a * Values within a row followed by the same letter are not s ignif icantly different at p = 0. 05. 76 0.65-0 2 4 6 8 PHOTOPERIOD REGIME (WKS) Figure 3.8. The September l i f t root dry weights of hemlock seedlings treated with variable weeks of short days. (Vertical bars indicate 1 standard error.) 77 (Table 3.10). A s l ight ly increasing amount of seedlings met the target standard with increasing weeks of short day treatment. 3.4.4 Root growth Capacity Short days did not affect the immediate root growth capacity of western hemlock seedlings (Table 3.11). However, significant differences between some treatments of Douglas-fir seedlings were demonstrated. The control, eight and six week regimes had similar RGC values, but the two and four week regimes exhibited signif icantly reduced RGC values compared to the eight week or control regimes. However, all values were above levels recommended for operational stock. 3.4.5 Frost Hardiness The frost hardiness tests for 17 August and 19 September were unsuccessful because of equipment malfunction. The freezing chest froze the seedlings approximately 7°C below the predetermined value. In addition, freezing was uneven throughout the freezer cavity. 3.5 Planting Trial Results 3.5.1 Climate Since the frost hardiness tests were unsuccessful, i t was important to record daily temperatures on the planting site immediately after outplanting. This could allow the inference of a natural frost testing providing frosts followed immediately after the outplanting. Daily minimum and maximum temperatures are reported in Table 3.12. Temperatures fe l l to 0°C or less on the second, f i f th and sixth night after the last day of 78 Table 3.12. Daily maximum and minimum temperatures and rainfal l from the last day of planting on 26 September 1983 until 31 October 1983. DATE DAYS FROM RAINFALL MAXIMUM MINIMUM PLANTING TEMPERATURE TEMPERATURE cm °C °C Sept.26 0 2.0 14 2 27 1 _ 13 2 28 2 - 14 0 29 3 - 13 1 30 4 12 3 Oct. 1 5 _ 9 0 2 6 1.0 13 1 3 7 4.0 14 -0.5 4 8 - 12 0 5 9 - 13 2 6 10 - 13 4.5 7 11 - 15 4.5 8 12 - 14 3 9 13 5.0 11 4.5 10 14 - 9 1 11 15 _ 11 0 12 16 - 16 -3 13 17 - 11 3.5 14 18 4 1 15 19 _ 3 -1 16 20 _ 7 -2 17 21 2.1 6 -1 18 22 2.0 8 3 19 23 15.0 6.5 -1 20 24 _ 5 0 21 25 18.0 10 -1 22 26 8 -1 23 27 6 0 24 28 38.0 1.5 1 25 29 _ 6 1 26 30 3.0 6.5 2 27 31 5 _ 28 32 _ _ 29 33 _ _ 30 34 28.0 31 35 79 planting. Repeated nighttime freezing occurred throughout October until logist ics prohibited further measurements at the end of the month. Rainfall was 118 cm from the completion of planting on September 26 until October 31. 3.5.3 Visual Observations Because of the occurrence of frosts immediately after outplanting, f ie ld observations of possible frost damage were attempted in early November, However, snowfall prohibited road access to the s i te . The f i r s t visual inspection of the planting t r i a l was not possible until early May once the snowpack was gone and crew personel were available. Thus, i t was not possible to speci f ica l ly conclude that any visual damage was due to i n i t i a l frost tolerance differences among treatments. Visual damage inspection involved subjective assessment of the needle, buds and tops without destroying any l ive seedlings. Damage was assessed while conducting a spring survival survey. In western hemlock winter topki l l occurred in 23% of the control seedlings (Table 3.13). The top foliage was deep red while lower foliage remained green. The frequency decreased with the increasing weeks of short days the seedlings received in the nursery. There was no topki l l in hemlock seedlings preconditioned with eight weeks of eight hour days. Since the seedlings were flushing during the survey period, missing and dead terminal buds were easily detected. The six and eight week photoregime treatments a l l had flushing terminal buds on the l ive trees. Live control plants had 21% of their terminal buds missing or dead while the two and four week regimes respectively had 4 and 3 percent terminal bud loss. Poor retention of one year needles was evident throughout a l l treatments. 80 Table 3.13. The incidence of top k i l l , dead or missing terminal buds and needle loss in outplanted western hemlock seedlings. Visual damage was observed in the spring following the fa l l planting. PHOTOREGIME (weeks) DAMAGE Topkill No Terminal Bud Needle Loss No. Live Trees Control 23 21 33 76 Two 9 4 57 83 Four 4 3 49 85 Six 3 0 33 88 Eight 0 0 33 90 Table 3.14. The incidence of top k i l l , dead or missing terminal buds and needle loss in outplanted Douglas-fir seedlings. Visual damage was observed in the spring following the fa l l planting. PHOTOREGIME DAMAGE {%)  (weeks) Topkill No Terminal Bud Needle Loss No. Live Trees Control 17 6 5 188 Two 7 4 6 18 0 Four 3 0 10 198 Six 2 2 5 189 Eight 3 3 7 191 81 The occurrence of these types of damage was not related to the inside or outside conditioning a l l treatments received prior to fa l l planting. A similar trend in topki l l incidence occurred in the Douglas-fir seedlings (Table 3.14). There were 17% dead tops in control plants, 1% in the two week regime and 3% in the eight week treatment. The occurrence of missing or dead terminal buds was not as prevalent in Douglas-fir. Even the control seedlings only had 8% of this type of damage. It decreased s l ight ly with increased weeks of short day preconditioning. Poor needle retention ranged from 4% to 10% throughout a l l treatments. No effect of the outside or inside conditioning was evident. Most of the one year foliage in a l l treatments was a lacklustre pale red green colour. The general discoloration of the one year needles in the Douglas-fir seedlings and the poor retention in both species possibly reflects the effects of winter desiccation. 3.5.3. Survival and Growth Performance There was l i t t l e difference between survival assessed in the spring and f a l l . Very l i t t l e mortality occurred in the f i r s t growing season. Only the 1984 fa l l survival results are discussed. Survival of western hemlock seedlings generally improved with increasing weeks of short day preconditioning (Figure 3.9). In an analysis of variance, however, only the eight week regime had signif icantly better survival (91%) than the control treatment (76%) (Table 3.15). This trend was not evident for Douglas-fir where survival was high throughout a l l treatments (Figure 3.10). It ranged from 89% in the two week interval to 98% in the four week regime (Table 3.16). 82 100 n 95-PHOTOPERIOD REGIME (WKS) Figure 3 . 9 . One year survival in outplanted western hemlock seedlinas treated with variable weeks of short days. 83 100 0 2 4 6 8 PHOTOPERIOD REGIME (WKS) Figure 3.10. One year survival of Douglas-fir seedlings treated with variable weeks of short days. 84 Figure 3.11. Total height, after one year, of outplanted western hemlock seedlings treated with variable weeks of short days. 85 2 4 6 PHOTOPERIOD REGIME (WKS) 8 Figure 3.12. Total height, after one year, of outplanted Douglas-fir seedlings treated with variable weeks of short days. (Vertical bars indicate standard error of the mean.) - Table 3.15. Survival results for western hemlock one year after planting. TREATMENT SURVIVAL [%) Control Two weeks Four weeks Six weeks Eight weeks * Values followed by the same letter are not s ignif icantly different at p = 0.05. 76' 86 86 88 ab ab ab 91' Table 3.16. Survival results for Douglas-fir one year after planting. TREATMENT SURVIVAL {%) Control Two weeks Four weeks Six weeks Eight weeks 9 0" ,ab* 89L 982 95 94 ab ab * Values followed by the same letter are not s ignif icantly di fferent at p = 0. 05. 87 Trends in total height of outplanted western hemlock and Douglas-fir seedlings are shown in Figures 3.11 and 3.12. Total height generally declined with increasing photoperiod because of the effect of the staggered arrangement of treatment in i t ia t ion dates on seedling height in the nursery. However, only the two and four week regimes had significant differences in total height of the western hemlock plants (Table 3.17). In Douglas-fir, the six and eight week treatments were s t i l l s ignif icantly less than the four week interval which were a l l s ignif icantly lower than the control and two week dormancy induction regimes (Table 3.18). Although total height declined with increasing treatment, the actual height increment in the f i r s t season of outplanting generally increased as the number of weeks of short day preconditioning increased (Figure 3.13 and 3.14). In fact height increment was 10.4 cm and 9.8 cm for the six and eight week regimes in western hemlock. This was s ignif icantly greater than the four week, two week or control seedling height increment which were respectively 7.1, 7.0 and 6.1 cm (Table 3.17). In Douglas-fir, significant differences in increment were also demonstrated between the control treatment and the six and eight week short day regimes (Table 3.18). The short day treatments were not s ignificantly different, although increment increased from 7.0 cm for the two week interval to 8.6 cm in the eight week regime. By calculating relative height growth rate (RGHR) height increment was corrected to allow for i n i t i a l size differences at the time of planting. In western hemlock, RGHR increased with increasing short day preconditioning (Figure 3.15). S t a t i s t i ca l ly , significant differences were the same as actual height increment differences (Table 3.15). A similar trend was found 88 Table 3 . 1 7 . Morphology measurements in outp lanted western hemlock. Seed l ings were assessed a f t e r one growing season i n October 1984. MEASUREMENTS PHOTOREGIME Cont ro l Two Weeks Four Weeks Six Weeks E ight Weeks Height (cm) 2 3 . 2 a b * 2 5 . 4 a 2 1 . 8 b 2 4 . 0 a b 2 1 . 8 b Height increment (cm) 6 . 1 b 7 . 0 b 7 . 1 b 1 0 . 4 a 9 . 8 a C a l i p e r 3 . 2 7 a b 3 . 4 1 a 2 . 9 9 b 3 . 4 8 a 3 . 3 5 a b Rhgr (yr ) 0.321° 0.350° 0.398° 0 . 5 8 5 a 0 .607 3 •Values w i t h i n rows f o l l o w e d by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t at p = 0. 05. ** R e l a t i v e he ight growth r a t e . Table 3 . 1 8 . Morphology measurements in outp lanted D o u g l a s - f i r . Seed l ings were assessed a f t e r one growing season i n October 1984. MEASUREMENTS PHOTOREGIME Cont ro l Two Weeks Four Weeks S ix Weeks E ight Weeks Height (cm) 2 7 . 0 a * 2 6 . 5 a 2 4 . 2 b 2 0 . 5 C 2 0 . 4 C Height increment (cm) 6.4° 7 . 5 a b 7 . 6 a b 8 . 0 a 8 . 6 a C a l i p e r 4 . 5 4 a 4 . 2 9 a 4 . 5 1 b 4 . 3 4 a 4 . 0 2 a Rhgr**(yr" ) 0 . 2 9 4 c 0 . 3 4 7 b c 0 . 3 8 8 b 0 . 5 1 1 3 0 . 5 2 3 3 •Values w i t h i n rows f o l l o w e d by the same l e t t e r are not s i g n i f i c a n t l y di f f e r e n t at p = 0. 05. ¥ ¥ 1 i 1 1 i 0 2 4 6 8 PHOTOPERIOD REGIME (WKS) Figure 3.13. Height increment after one growing season in western hemlock seedlings treated with variable weeks of short days. (Vertical bars indicate standard error of the mean.) 0 2 4 6 8 PHOTOPERIOD REGIME (WKS) Figure 3.14. Height increment after one growing season in Douglas-fir seedlings treated with variable weeks of short days. (Vertical bars indicate standard error of the mean.) 91 0 . 6 5 0 55 O rr r -I e> LU I LLI > 0 . 4 5 -0 . 3 5 0 25 Figure 3.15. 2 4 6 PHOTOPERIOD REGIME (WKS) Relative height growth (yr~ ) after one growing season in western hemlock seedlings treated with variable weeks of short days. (Vertical bars indicate standard error of the mean.) 92 0.65 0.55-0.25 J 1 1 ; 1 ' 0 2 4 6 8 PHOTOPERIOD REGIME (WKS) Figure 3.16. Relative height growth (yr ) of outplanted Douglas-fir seedlings treated with variable weeks of short days. (Vertical bars indicate standard error of the mean.) 93 for Douglas-fir (Figure 3.16). However, significant differences between short day treatments were established. RGHR of the six and eight week regimes were s ignif icantly greater than those of the four and two week regimes as well as the control. The four week treatment had a s ig i f icant ly higher RGHR than the control. Caliper of either species was unaffected by short day pre-conditioning. The inside or outside conditioning which followed the short day treatments in the nursery did not influence any of these measured growth characteristics. 3.6 Discussion 3.6.1 Nursery Tria l Height growth in Douglas-fir and western hemlock container seedlings was successfully controlled by in i t ia t ing early budset with the application of short days. Buds formed in two to three weeks in Douglas-fir and three to four weeks in western hemlock. The rate of bud formation may vary s l ightly from year to year in CIP Forest Products nursery because the rate of budset in Douglas-fir apparently changed with the date of treatment i n i t i a t i o n . However, buds possibly may have formed faster in later init iated treatments because of the inductive effect of the naturally declining photoperiod. Twenty percent of the plants under a natural photoperiod had set bud when the photoregimes were completed on 17 August. This photoperiodic effect seems l ike ly because date of treatment in i t ia t ion did not affect bud formation in western hemlock and the control hemlock seedling 94 did not have terminal buds on 17 August. Cheung (1973) also demonstrated that when short days were applied to western hemlock in the 14th, 16th and 18th week from germination, formation of terminal buds was not influenced by time of application. Temperature, however, is a major factor which affects bud formation. Cheung (1978) reported that under an eight hour regime bud formation in western hemlock was slower at temperatures above or below 20 °C . Nelson and Lavender (1976) suggested that warm temperatures of 25°C and 20°C in a short photoperiod induced dormancy in western hemlock at a faster rate than a cool thermoperiod. In Douglas-fir, Lavender and Overton (1972) reported that cool temperature delayed dormancy in coastal Douglas-fir seedlings maintained under a nine hour photoperiod. In general, i t would appear that budset is quickly init iated under a short day regime with warm temperatures. The effect of temperature on bud formation must be considered when drawing conclusions from this study. Daily maximum temperatures averaged 26°C ranging from 17°C to 3 2 ° C . The summer of 1983 was only moderately warm and dry for southern Vancouver Island. A hotter or cooler summer may affect rate of bud formation and hence, the amount of shoot extension once treatment is in i t i a ted . Short days generally reduced shoot dry weight in Douglas-fir and western hemlock. Root dry weight was unaffected in both species when the photoregimes were just completed on 17 August. Similar results are reported in the l i terature . Short photoperiod greatly reduced shoot dry weight accumulation in black spruce, but i t only s l ightly affected root dry weight (D'Aooust and Cameron 1981). However, Cheung (1973) demonstrated that short days significantly reduced shoot and root dry weight accumulations in western 95 hemlock seedlings. The root/shoot dry weight ratio increased, implying the root system was less affected by short photoperiod than the shoot. The freshweight of Douglas-fir seedlings also declined under a short photoperiod (Lavender and Hermann 1970). In addition, the roots were also affected by short days since the number of active roots decreased. In a second study, Lavender and Wareing (1972) also showed that the number of active roots declined when Douglas-fir was pretreated with short days. Unlike these reports, root biomass and root growth were not reduced by short day pretreatments in this thesis study. Douglas-fir root dry weight was similar throughout a l l treatments at both sample periods. In addition, the six and eight week regimes had similar root growth capacity values to that of control. Although not s t a t i s t i ca l ly s ignif icant, western hemlock root dry weight actually increased in the six and eight week regimes compared to a l l other treatments after the four week conditioning period. In addition, the proportion of seedlings exceeding the MOF cull standard for root dry weight dramatically increased with increasing weeks of short day treatment. This occurrence reflects the fa l l surge in root growth which commonly occurs once coniferous species set bud. Budset was complete in the four, six and eight week regimes. Hence once the daylength was extended to a natural period, increased photosynthates were probably channelled to the roots. In the treatment where budset was incomplete, photosynthates were probably ut i l ized to a greater extent in the shoot. Hence, root biomass was s l ightly less in the control and two week photoregimes. This theory seems plausible because 96 the RGC test indicates that the number of active roots was not reduced in hemlock seedlings pretreated with short days. Hence, the expression of potential root growth probably varied among treatments because of the varying competition for photosynthates in seedlings with complete budset or active extending apices. A similar trend was not apparent in Douglas-fir because incidence of budset was high throughout a l l treatments. That i s , the majority of shoots in a l l treatments were no longer elongating. 3.6.2 Planting Tria l Preconditoning with short days generally improved one year survival and outplanting performance of fa l l planted western hemlock and Douglas-fir container stock. The increased survival of the treated western hemlock seedlings compared to hemlock controls most l ike ly reflects the increased frost tolerance associated with short day preconditioning especially since frost occurred within the f i r s t week following outplanting. The type of visual damage to the seedlings also supports this claim. The high incidence of topkil l in the control and two week photoregimes and the high incidence of budkill in the control seedlings suggest that the stems and buds were not as tolerant of the early fa l l frosts and winter desiccation. That i s , the seedlings from these treatments were less hardened off at the time of the fa l l l i f t and outplanting. Stem tissue was probably more succulent and more susceptible to damage by the frosts which immediately followed the outplanting. A difference in stress resistance or frost tolerance in the control and treated hemlock seedlings can be inferred from the survival data. The higher mortality of the control plants suggests stress resistance or 97 possibly frost hardiness was lower than the seedlings preconditioned with short days. Budset occurred in the control treatment during the end of the four weeks of conditioning period. Thus the hardening off phase which commonly followed budset had not yet developed by the time the seedlings were l i f t e d . Because these plants had only just entered into dormancy, resistance to the desiccating effects of l i f t i n g , handling and planting was possibly lower than a l l other treatments where budset occurred a minimum of four weeks prior to l i f t i n g . Thus, besides lower frost tolerance, a reduced resistance to the stress associated with planting may also account for the lower survival of the control treatment. It is unfortunate that the frost tolerance tests for the 19 August and 19 September sample periods were unsuccessful because the results would have permitted a more conclusive explanation for why survival improved and seedling damage decreased with increasing weeks of short day preconditioning of western hemlock seedlings. However, there is conclusive evidence reported in the literature which shows that preconditioning with short days enhances frost hardiness in several coniferous species. Frost hardiness was enhanced in Douglas-fir seedlings under an eight week regime of eight and ten hour days (McCreary et a l . 1978). Plants grown under natural and extended photoperiods were less hardy. Although a six hour short day improved frost hardiness compared to the control treatment, i t reduced hardiness levels compared to the eight and ten hour regimes. The importance of a conditioning period following short day treatment was also demonstrated whereby the seedlings preconditioned with short days continued to develop greater levels of frost hardiness up to four 98 weeks later . Timmis and Worrall (1975) also demonstrated that a short day of eight or ten hours induced frost hardiness in Douglas-fir. Hardiness levels were again s l ight ly reduced by a six hour regime. Tanaka (1974) reported that cold hardiness development was accelerated by two or three weeks in Douglas-fir container seedlings preconditioned with eight hour days. This is an important finding for stock scheduled for early fa l l l i f t and outplanting. van den Driessche (197 0) reported that frost hardiness f i r s t increased at the end of the fourth week of an eight hour photoregime, although the rate of hardening was affected by the level of l ight intensity. After eight weeks of treatment, seedlings cultured under an eight hour regime were s ignif icantly hardier than plants grown in a sixteen hour photoperiod, providing the l ight intensity was sufficient to meet photosynthesis requirements. However, hardiness levels of seedlings in long and short day photoregimes were similar several weeks later . In a second study, van den Driessche (1969b) also demonstrated that short days enhanced hardiness in the f a l l , but by mid-November seedlings grown in a natural photoperiod had attained similar levels of cold hardiness. Thus, for a coastal species an operational advantage of short day preconditioning is accelerated frost hardiness in stock scheduled for high elevation sites in the f a l l . In addition to frost hardiness benefits, Lavender and Wareing (1973) demonstrated that short day preconditioning of Douglas-fir enhanced seedling resistance to the effects of root pruning, l i f t i n g and cold storage. This documented finding may account for the poorer survival of the hemlock control seedlings. These plants possibly had not yet developed resistance to the stresses of l i f t i n g and planting. 99 The effect of short days on accelerated cold acclimation has been demonstrated in other species. After six weeks of eight hour days at 2 0 ° C , black and white spruce seedlings were hardy to at least - 9 ° C , the l imit of the frost evaluation procedure employed in the study (Columbo et a l . 1981). In a fa l l outplanting of Norway spruce, frost injury to the needles was reduced from 63% to 5% with only a two week short day preconditioning period in July or August (Sandvik 1980). Several other studies document the positive effects of short days on frost hardiness of Norway spruce and Scots pine (Aronsson 1975; Christersson 1978; McGuire and Flint 1962; Rosvall-Ahnebrink 1981). There is not, however, much l iterature on the frost hardiness of coastal western hemlock. Timmis (1976) reported that short days and cool temperatures promote cold acclimation in western hemlock as i t generally does in northern coniferous species. Cheung (1978) examined frost hardiness of high and low elevation western hemlock seedlings under three temperature regimes of 1 0 ° , 20° and 30°C and two photoperiods of sixteen or eight hours. Frost hardiness was greatest under the short day regime of 10 °C , but bud formation was inhibited. Consequently a short day regime of 20°C was recommended as a dormancy induction technique. Other studies on photoperiod in western hemlock have dealt principally with dormancy, c h i l l i n g requirement and growth response, but not frost hardiness (Hauessler 1981; Nelson and Lavender 1976). From this brief discussion of the l iterature i t appears there is much evidence to support the inference that differences in survival and seedling stem and bud damage in the western hemlock seedlings reflect differences in frost hardiness or overall stress resistance levels produced 100 by the treatments of natural photoperiod or short days. When Norway spruce seedlings were preconditioned with four weeks of 8 hour days and outplanted in the f a l l , frost damage was reduced and three year survival was improved (Rosvall-Ahnebrink 1981). Survival of fa l l planted black spruce also increased with short day pretreatment (D'Aoust and Cameron 19819). Tanaka (1974) reported similar results when Douglas-fir container seedlings were fa l l planted on high elevation s i tes . In a l l these outplanting studies the improved survival was attributed to enhanced frost hardiness promoted by short day preconditioning. Although western hemlock survival increased with increasing weeks of short days, only the eight week photoregime had signif icantly higher survival than the control . Nonetheless, even the two week photoregime had 10% higher survival than the control or natural photoperiod treatment. Thus, the importance of early budset prior to l i f t i n g and the effect of short day treatment on fa l l survival of fa l l planted hemlock was demonstrated. The drastic reduction in topki l l or frost damage between natural photoperiod and any of the short day treatments was also shown. The trend in survival with short day treatment was not so apparent in the Douglas-fir seedlings. Survival was higher in the four, six and eight week photoregimes compared to the natural photoperiod or the two week short day regime, but i t was not s t a t i s t i ca l ly s ignif icant. Interestingly, even these treatments had a high rate of survival at 90% and 89% respectively. This high survival of these treatments probably occurred because over 20% of the control or natural photoperiod and 80% of the two week regime had set bud when the photoregimes were completed on 19 August 1984. Thus, even the 101 control seedlings underwent a hardening off period before the l i f t on 19 September. Thus, some degree of frost hardiness and stress resistance had probably developed when the seedlings were outplanted. The early budset of the control plants implies that the natural photoperiod had declined below the level c r i t i c a l for shoot elongation for this particular provenance of Douglas-fir or that the irr igat ion regime was insufficient to maintain growth. Since hemlock shoots were s t i l l elongating under a natural photoperiod, i t would suggest that the irr igat ion was frequent enough to maintain growth and that the c r i t i c a l photoperiod for the Douglas-fir seedlot was greater than the c r i t i c a l daylength for the hemlock seedlot. In spite of the fact that buds had formed throughout a l l of the Douglas-fir treatments, short day pretreatment s t i l l resulted in some differences in survival and seedling frost damage. This suggests seedlings pretreated with more than two weeks of eight hour days had attained a s l ightly higher level of frost hardiness. Topk i l l , an indication of frost damage to the stem, was s t i l l higher in the control with an incidence of 17%.. There was no difference in topki l l between the four, six and eight week regimes which suggest that frost hardiness in the stem was similar for these treatments. The s l ight ly higher survival in these three photoregimes also implies a short day preconditioning period greater than two weeks enhanced overall frost hardiness or stress resistance. The hemlock seedlings generally exhibited s l ightly lower survival at each treatment level than the Douglas-fir plants. One possible explanation is that frost hardiness or stress resistance was higher in the Douglas-fir during l i f t i n g and planting. Timmis (1976) suggested that 102 hemlock acclimates slower than Douglas-fir and speculated that hemlock was a less desirable species for fa l l planting. Secondly, the study site may have been more suitable for Douglas-fir. Pretreatment with short days significantly increased f i r s t year growth on an absolute and relative basis of fa l l planted western hemlock and Douglas-fir seedlings. In western hemlock a preconditioning period of six or eight weeks produced a s ignif icantly greater height growth response. In Douglas-fir, the six and eight week treatments had signif icantly greater height growth compared to the control but not to the two and four week regimes. However, relative height growth was significantly greater in the six and eight week photoregimes compared to a l l other treatments. Few of the published studies on photoperiodism and dormancy have discussed one year height growth in a planting t r i a l . Rosvall-Ahnebrink (1981) demonstrated that Scots pine seedlings and Norway spruce seedlings treated with four weeks of short days, had greater height growth during the f i r s t growing season. In addition, pretreatment with short days also resulted in earl ier budflush in the f i r s t spring and delayed apical growth cessation in the f a l l . This probably accounted for the increased shoot elongation throughout the f i r s t growing season. Under a controlled environment, Lavender and Wareing (1970) reported that short day pretreatment accelerated bud act ivi ty in Douglas-fir seedlings compared to a long day photoregime. A similar result was reported for western hemlock (Nelson and Lavender 1976). Short day preconditioning also accelerated budflush in Norway spruce seedlings maintained in an environment chamber (Sandvik 1980). However, Sandvik (1980) attributed the earl ier budflush to the higher fo l iar nitrogen reserves that accumulated in 103 the fa l l directly after treatment with short days. Cheung (1973) also discovered that fo l iar nitrogen increased in western hemlock seedlings treated with eight hour days compared to sixteen hours or a moisture stress regime. In addition to greater fo l iar nitrogen accumulation and an alteration in growth pattern, a third explanation to possibly account for increased height growth can be inferred from Colombo et a l . (1981). Apical meristems were larger and contained far more needle primordia in spruce seedlings grown under an eight hour day at 2 0 ° C . However, these seedlings were compared to production run stock where temperatures were not controlled. That i s , cooler tempeatures may also account for the smaller bud size with fewer primordia. Since the timing of budflush and analysis of fo l iar nitrogen was not assessed in this study i t is not possible to infer an explanation for increased height growth from the data. However, bud act ivi ty was assessed in seedlings grown under a natural photoperiod and in an eight hour regime in Study II. These results are reviewed in the next chapter. Nonetheless, i t was clearly demonstrated that a short day treatment of six or eight weeks increased f i r s t year growth performance of fa l l planted western hemlock and Douglas-fir container seedlings. Potential for this may be found in the selection and proper scheduling of a dormancy induction regime that enhances frost hardiness, stress tolerance and growth potential of operational nursery stock. Although such a regime cannot be recommended from this study, the potential benefits of short days were demonstrated. 104 3.7 Conclusions Short photoperiod applied to Douglas-fir and western hemlock container stock quickly ini t iated homogeneous budset. It was successfully demonstrated that short days was an effective operational tool to control or stop height growth in order to meet predetermined target specifications for seedlings. Shoot elongation in both species generally stopped in about three weeks when treatments were init iated in late June and early July and in two weeks when treatments were started in late July or early August. The averageheight growth after the short photoperiod was init iated was 3.7 cm in Douglas-fir and 4.2 cm in western hemlock. Douglas-fir buds formed in three to four weeks in the treatments init iated in late June and early July and two to three weeks in later ini t ia ted treatments. The faster rate of bud formation was attributed to dormancy induction effect of a naturally declining photoperiod in this particular Douglas-fir provenance. Buds were not detected on western hemlock seedlings until the fourth week of the short photoregime. It should be pointed out that the rate of apical growth cessation and bud formation is specific to the environmental conditions of this particular study. Incidence of second flushing in seedlings pretreated with short days was nonexistant in western hemlock and only 1% in Douglas-fir. Caliper was unaffected by short day pretreatment. Shoot dry weight was s ignif icantly reduced by increasing weeks of short day treatment. However, this reduction in shoot dry weight reflects the effect of staggered treatment in i t i a t ion dates. The eight week treatment was init iated in late 105 June when the seedlings were shorter than when four weeks were started in late July. Outdoor conditioning enhanced shoot dry weight accumulation in both species. Short day pretreatments did not adversely affect root dry weight or root growth capacity. In fact, the fa l l surge in root growth in western hemlock seedlings was accelerated in the six and eight week photoregimes. Only 65, 65 and 60%, respectively of the control, two week and four week short day seedlings had root dry weights which exceeded the MOF cull standard while 90% and 100% of the six and eight week treatments exceeded this standard at the September l i f t . Once again this result possibly reflects the importance of early budset in i t i a t ion to encourage fa l l root growth prior to l i f t i n g for a fa l l scheduled outplanting. That i s , i t is d i f f i cu l t to discern whether the six and eight week regimes enhanced root growth or the early init iated date accounted for this surge in root growth. This trend was not evident in the Douglas-fir experiment where even control seedlings had set bud three to four weeks prior to l i f t . This implies that the early in i t ia t ion of budset in the hemlock seedlings led to increased root growth in September. Nothing can be concluded about frost hardiness levels at the time of photoregime completion and after a four week conditioning period because the tests were unsuccessful. However, inferences about frost hardiness levels produced by the different treatments can be drawn from the fa l l planting t r i a l . Winter topki l l is the result of frost damage to seedling shoots. Incidence of topki l l was 23% in the hemlock control seedlings, and 9% in the two week plants. It decreased to 0% in the eight week seedlings. 106 In addition, terminal bud k i l l was 21% in the control compared to 4% in the four week and 0% in the six and eight week seedlings. Incidence of frost damage to western hemlock plants declined with increasing weeks of short day treatment. However, damage frequency was similar for the six and eight week regimes. These results imply that short day pretreatment of six or eight weeks enhanced frost tolerance to levels greater than seedlings grown under a natural summer photoperiod. The preconditioning of Douglas-fir seedlings with short photoperiod also enhanced frost hardiness levels . Topkill was 17% and 7% respectively inthe control and two week plants. The incidence was only 2 or 3% in the four, six and eight week treatments. Short day pretreatment also enhanced the planting survival of western hemlock seedlings. Survival increased with increasing weeks of short days. However, only the eight week regime was s ignif icantly higher. A treatment difference in stress resistance to the desiccating effects of l i f t i n g , handling and planting or in frost hardiness can be inferred from the data. Survival of Douglas-fir seedlings increased s l i ght ly , but not s ignif icantly when more than two weeks of short days were applied. There were no s tat i s t ica l differences between most of the treatments. However, based on incidence of frost damage and the slight improvement in survival , short day pretreatment greater than two weeks enhanced frost hardiness in the Douglas-fir seedlings. Short photoperiod signif icantly improved growth performance of fa l l planted Douglas-fir and western hemlock container seedlings. In both species, the six and eight week regimes produced a s ignif icantly greater 107 height growth response than a l l other treatments. The western hemlock treated with six week regimes grew an average of 10.4 cm compared to 6.1 in the plants under a natural photoperiod, an increase of 4.3 cm. The increase in height growth was less dramatic for Douglas-fir. Control plants grew 6.4 cm while the eight week seedlings grew 8.6 cm, an increase of 2.2 cm. Caliper growth was unaffected by short day precondition. Based on plantation growth performance, survival rates and incidence of frost damage, a six or eight week photoregime appeared to produce the best quality western hemlock seedlings for this study. It was also shown that a short photoperiod of this duration did not adversely affect root biomass or root growth capacity. Selection of the best photoregime for Douglas-fir is not so obvious since even the control seedlings set early buds. However, based on the outplanting results , i t would appear that at least four weeks of short days were necessary to grow a seedling that survived and grew better on this particular planting s i te . A few points must be considered when reviewing these conclusions. The outplanting t r i a l was conducted on only one site type, that was assessed as having a poor to medium productivity. Growth performance and survival will vary according to productivity, aspect, elevation, moisture regime and various other factors. In addition, the t r i a l involves only one summer which may or may not reflect weather conditions typical of south Vancouver Island. It would also be of interest to examine survival and growth after the second and third growing season. If CIP Forest Product employs short days as an operational dormancy induction, plantations should be monitored for survival and growth performance. 108 CHAPTER FOUR STUDY TWO DORMANCY INDUCTION OF DOUGLAS-FIR CONTAINERIZED SEEDLINGS: A COMPARISON BETWEEN MOISTURE STRESS, SHORT DAYS AND A COMBINATION OF MOISTURE STRESS FOLLOWED BY SHORT DAYS 4.1 Introduction The 1983 operational t r i a l demonstrated the effectiveness of short days in in i t ia t ing budset, enhancing survival in western hemlock and f i r s t year growth performance in hemlock and Douglas-fir container seedlings. There i s , however, evidence that moisture stress is also an effective tool in controlling height and inducing dormancy in forest seedlings (Blake et a l . 1979; Cheung 1973; Lavender and Cleary 1974; McDonald and Running 1979; Timmis and Tanaka 1976; Zaerr et a l . 1981). Several studies indicate that short days quickly enhance frost hardiness in Douglas-fir (McCreary et a l . 1978; Tanaka 1974; Timmis and Worrall 1975; van den Driessche 1969b, 1970). Reports on the effect of moisture stress on frost hardiness are conf l ic t ing . Blake et a l . (1979) demonstrated that hardiness was enhanced at a shoot water potential of -5 to -10 bars and reduced at a plant moisture stress of -10 to -15 bars. Timmis and Tanaka (1976) reported that a stress of -6 bars enhanced hardiness compared to -12 bars. Van den Driessche (1969b) concluded that moisture stress had no significant effect on frost hardiness in Douglas-fir seedlings; but under a short photoperiod seedlings regularly watered acclimated faster than seedlings infrequently irr igated. In a l l these studies frost hardiness tests were conducted immediately after treatment or within 11.5 weeks of treatment. Frost hardiness is an important attribute of seedling quality. It enables seedlings to withstand the freezing temperatures of outdoor 109 overwintering or of winter l i f t i n g and cold storage. Selection of an operational dormancy induction technique should enhance or at least maintain hardiness levels . Other measureable attributes to consider in developing an operational tool for controlling height growth, and in i t i a t ing and maintaining budset are root growth capacity, dormancy intensity and morphological characteristics such as bud height and root dry weight. Extensive evidence indicates that moisture stress and short days are both effective in inducing budset. An operational t r i a l was conducted to assess the effectiveness of both as operational induction tools for western hemlock and Douglas-fir. The study was designed to evaluate and compare the effects of short days and moisture stress on the physiological and morphological quality of Douglas-fir container seedlings. The physiological parameters included root growth capacity, dormancy intensity and frost hardiness at the time of an operational January l i f t , and root growth capacity and dormancy intensity after five weeks of operational cold storage. 4.2 Methods Four levels of moisture stress and two photoperiod regimes were factorial ly applied to Douglas-fir seedlings in a completely randomized design. The coastal provenance seedlings were from seedlot 7276 collected from seedzone 1020 at an elevation of 925 m. They were sown in 313 styroblock containers on 23 March 1984 at the CIP Forest Products Nursery in Saanichton, Brit i sh Columbia. They were cultured under the nursery's standard f e r t i l i z e r and irr igat ion regimes until treatments commenced on 19 July 1984 (Appendix III). The four levels of moisture stress were: control , 110 l ight , medium and severe which respectively corresponded to predawn pressure bomb readings of 0 to -5 bars, -10 bars, -15 bars and -25 bars. Styroblock weights and soil water content were measured concurrently in order to correlate them with pressure bomb readings. Soil water content was determined by bulking the planting media from the pressure bombed seedlings. Soil samples were weighed immediately after pressure bombing and again after approximately 24 hours of oven drying at 7 0°C. Soil water content was estimated as a percentage of the oven dried weight of s o i l . The stress period was conducted over a two week period. Each time seedlings reached their respective stress levels , they were rewatered and allowed to dry out agai n. The two levels of photoperiod were the natural daylength and four weeks of eight hour days. The photoperiod was controlled in a greenhouse outfitted with black plastic curtains. Light leaks were monitored on the treatment benches with a LICOR photometer. Temperatures were recorded by a hygrothermograph. Although moisture stress and photoperiod regimes were factor ia l ly arranged, not a l l treatments were applied concurrently. The four levels of moisture stress and the unstressed short photoperiod treatments commenced on 19 July. Once the two weeks of stress were completed, half of a l l these treatments then received four weeks of eight hour days until 3 September. When a l l treatments were finished, a l l seedlings were cultured under CIP Forest Products Nursery standard regimes. The seedlings were l i f t ed in the third week of January 1985 and placed into cold storage for five weeks. I l l 4.2.2 Measurements Treatment effects were evaluated by assessing seedling morphology, root growth capacity, frost hardiness and dormancy intensity. Height and caliper were measured weekly during the treatment period. Shoot and root dry weights were recorded every second week. Once the treatments were completed, a l l measurements were done monthly until the seedlings were l i f t ed in January. Forty-five seedlings per treatment were measured for each sample period. Root growth capacity (RGC) was tested at the time of seedling l i f t and after five weeks of cold storage. The RGC tests were conducted according to the procedures outlined by the Ministry of Forest (Appendix 1). Twenty trees were sampled from each treatment at each test period. Frost hardiness was assessed by subjecting seedlings to a range of freezing temperatures and determining the temperature at which 50% (LT 5 0 ) of the seedlings died or were severely damaged. Bud, stem and needle damage were a l l included in the assessment. Eighty seedlings from each treatment were shipped to a laboratory in Washington state in early January. The procedures for this test are outlined in Appendix II. Dormancy intensity was simply assessed by placing twenty sample seedlings per treatment into a growth chamber and monitoring the number of days required for budbreak. The environment conditions in*the chamber were 20°C during the day and night with a daylength of 16 hours. 4.2.3 Statist ical Analysis Treatment effects were assessed in analyses of variance. When either or both of the two factors were s ignif icant , treatment means were compared by 112 Newman-Keul's multiple range test . Several of the analyzed variables did not meet the assumption of homogeneous variance, as indicated by a Bartlett Chi-square test. Transformation did not result in homoescasdicity. Consequently, interpretations from these analyses should be made with caution. The analyses which did not meet this basic assumption are outlined in Appendix IV. Tables for analyses of variance are reported in Appendix V. 4.3 Results 4.3.1 Treatments The actual pressure bomb values for each level of moisture stress were similar to proposed levels (Table 4.1). Severe, medium, l ight and control moisture stress treatments corresponded to average predawn shoot water potential measurements of -23.4, -17.5, -9.5 and -4.9 bars, respectively. Daily pressure bomb readings and the ranges are included in Appendix VI. The stress period lasted sixteen days during which time each treatment reached its respective stress level at least twice. The block weights, water loss on a weight basis and soil water content associated with each class of moisture stress are reported in Table 4.1. It is important for operational nursery staff to note that an average 313 block weight of 4.5 kg produced a l ight stress of -9.7 bars while only a sl ight decrease to 4.35 kg resulted in a medium moisture stress of -17.5 bars. A similarly small reduction to 4.05 kg incurred a severe stress of -23.4 bars. The relationships between shoot water potential and styroblock weight, and plant moisture stress and soil water content are shown in Figures 4.1 and 4.2. From these graphs i t is evident that as soil water content or styroblock weights become increasingly lower, small changes in these measurements produce major changes in plant Table 4.1 Plant moisture stress, styroblock weight, waterloss on a weight basis, and soil water content at the time each treatment was watered. DAY OF STYROBLOCK WATER ACTUAL PROPOSED SOIL WATER TREATMENT IRRIGATION1 WEIGHT (kg) LOSS (kg) PMS (-bars) PMS (-bars) CONTENT {%) Control 1-PM 6.2 1.25 66 5-PM 5.7 2.35 - 60 8-AM 5.6 2.20 4.1 62 12-AM 5.1 2.90 5.7 58 16-AM 4.9 3. 00 5.0 56 Average 5.5 2.34 4.9 5.0 6 0.4 Light Stress 6-AM 4.5 3.10 9.6 41 11-AM 4.6 3.30 9.8 49 16-AM 4.5 3.00 9.0 41 Average 4.53 3.13 9.5 10.0 40.3 Medium Stress 7-AM 4.3 3.5 18.4 35 13-AM 4.4 3.10 16.7 33 Average 4.35 3.3 17.5 15.0 34 Severe Stress 8-AM 4.0 3.70 23.8 32 16-AM 4.1 3.70 23.0 29 Average 4.05 3.70 23.4 25.0 30.5 Number of days from treatment in i t i a t ion . 114 SOIL WATER CONTENT(%) Figure 4.1 The relationship between shoot water potential and soil water content (%). 115 Figure 4.2. The relationship between water potential and styroblock weight. 116 moisture stress. The curves represented in these figures are specific to the conditions of this study and cannot be used to estimate stress levels in Douglas-fir seedlings in another growing season. 4.3.2 Daily Climate The weather remained hot and dry throughout the dormancy induction period. During the actual moisture stress period from 19 July 1984 to 3 August 1984, daily maximum temperatures ranged from 23°. to 34°C for an average temperature of 2 9 . 5 ° C . Nightly minimum temperatures varied from 7 ° to 1 6 ° C , for an average temperature of 13 °C . The temperature regimes in the short day greenhouse were warmer. Daily temperatures rose as high as 38 °C . They were frequently in the mid-thirties throughout the entire treatment period. No l ight leaks were detected on the research bench in the short day greenhouse. 4.3.3 Bud Formation and Incidence of Reflushing In the f i r s t sixteen days of experimentation a short photoperiod was compared to four levels of moisture stress under a natural daylength. The rate of bud formation varied between treatments at the end of this approximate two week period (Table 4.2). Short days exhibited the fastest rate of bud formation where 87% of the seedlings set terminal buds in sixteen days. This was s ignif icantly greater than the control plants with 44% budset and the medium stress seedlings with 51% incidence. Although bud formation was highest under the short day regime, i t did not differ s ignif icantly from the l ight stress at 69% and the severe stress at 80%. With the exception of the medium class of moisture stress, increasing levels of moisture stress 117 resulted in an increasing rate of bud formation. However, this trend was not s t a t i s t i ca l ly s ignif icant. At the end of the moisture stress period, half of the blocks from each stress treatment were placed under a short photoperiod. Two weeks later or after four weeks from project i n i t i a t i o n , photoperiod and moisture stress pretreatment s ignif icantly affected incidence of budset. There was also a significant interaction between moisture stress preconditioning and photoperiod (Figure 4.3). The interaction occurred at the control or regular irr igat ion level of moisture stress where the magnitude of the response to a natural or short daylength was different from any other level of moisture stress. At each level of moisture stress, incidence of budset was s ignif icantly higher in the short day regime (Table 4.3). Under a natural photoperiod, budset incidence was s ignif icantly greater in seedlings treated with a l i gh t , medium or severe moisture stress compared to the control plants. Although pretreatment with moisture stress increased terminal bud formation, no significant differences were demonstrated between these three classes. In a short day regime, preconditioning with moisture stress did not s t a t i s t i ca l ly affect budset four weeks from project i n i t i a t i o n . That i s , after two weeks of short days, the moisture regime applied prior to short day treatment did not influence the number of seedlings with terminal buds. When the means of a l l treatment combinations were compared, seedlings under a short day regime had s ignif icantly more terminal buds after four weeks from project i n i t i a t i o n , regardless of moisture regime (Table 4.4). Only the short day seedlings pretreated with medium moisture stress were similar to the l ight and severe stressed plants under a natural daylength. 118 Table 4.2 Terminal bud formation in Douglas-fir seedlings maintained under five dormancy induction regimes. Assessment made sixteen days after treatment i n i t i a t i o n . TREATMENT PROPORTION OF SEEDLINGS WITH TERMINAL BUDS {%) Natural Photoperiod Control 44c Light Stress 69 a b be Medium Stress 51 Severe Stress 8 0a Short Photoperiod 87 a •Values followed by the same letter are not s ignif icantly different at p = 0.05. Table 4.3 The effect of photoperiod and moisture stress on terminal bud formation of Douglas-fir seedlings. Assessment made four weeks after project i n i t i a t i o n . PHOTOPERIOD MOISTURE STRESS Natural Short %seedlings with terminal buds Control l l . l 6 * 1 9 3 . 3 a I 1 a l * * a l l Light 62 .2 a i 8 8 . 9 3 1 1 Medium 51 .1 a I 8 0 . 0 a I 1 Severe 64 .4 a I 9 1 . 1 a I 1 •Values within a column followed by the same letter are. not s ignificantly different at p = 0.05. **Values within a row followed by the same Roman number are not s ignif icantly different at p = 0. 05. 119 Table 4.4 Bud formation and incidence of reflushing in eight dormancy induction treatments applied to Douglas-fir seedlings. TREATMENT WEEKS FROM TREATMENT INITIATION FOUR SIX %Budset %Reflushed %Budset %Reflushed Natural Photoperiod control d* 11 ° 82a 76b 24b light stress 6 2 b c 24b ioo a oa medium stress 51C 2 0b 98d 0a severe stress 64 b c 24b 98a O3 Short Photoperiod control 93 a 4 b 96a 4a l ight stress 89 a l l b 100a O3 medium stress 8 0 a b 13b ioo a o3 severe stess 91 a 9 b 100a o3 •Values within a column followed by the same letter are not s ignif icantly different at p = 0.05. 120 100 0 J , 1 i 1 — 1 Control Light Medium Severe MOISTURE REGIME Figure 4.3. Budset incidence in Douglas-fir seedlings after four weeks of treatment with moisture stress, short days or a combination of both. (Vertical bars indicate standard error of the mean.) 121 Figure 4.4. Incidence of flushed terminal buds in Douglas-f i r seedlings after four weeks of treatment with moisture stress and short days. (Vertical bars indicate standard error of the mean.) 122 For a l l levels of moisture stress, short photoperiod reduced the incidence of reflushing four weeks from treatment in i t ia t ion (Figure 4.4). However, the only significant difference between photoperiods occurred at the control moisture stress level (Table 4.4). That i s , moisture regime only affected the occurrence of reflushing under a natural photoperiod at the control moisture regime leve l . To summarize, after a four week period, a short photoperiod, regardless of the level of moisture stress, was more effective in in i t ia t ing and maintaining budset compared to seedlings under a natural photoperiod. Six weeks from treatment in i t i a t ion terminal buds had formed in 96% or more of the seedlings in a l l treatments but the control moisture regime in a natural daylength. Only 76% of these plants had terminal buds. Incidence of reflushing was 24% in this treatment compared to negligible levels in a l l ether treatment combinations (Table 4.4). Therefore, moisture stress, short days or a combination of both effectively init iated and maintained budset after a six week period commencing in the third week of July and ending in the f i r s t week of September. A natural photoperiod without moisture stress was least effective. 4.3.4 Morphology Seedlings maintained under a natural photoperiod with a control moisture regime were s ignif icantly larger in height, cal iper, shoot dry weight and root dry weight than a l l other treatment combinations at the time of the January l i f t (Table 4.5). Because of the size differences produced only under these particular conditions or this treatment combination, Table 4.5 Morphology measurements of Douglas-fir seedlings at the time of the January 1985 l i f t . MEASUREMENTS TREATMENT Height Caliper Ht/C1 Shoot Dry Root Dry S/R2 Bud Height (cm) (mm) weight (g) Weight (g) (mm) Natural Photoperiod control a* 23.3a 2.98a 7.8 1.74a 0.83C 2.1 5.3a l ight stress 17.4 c d e 2.47C 7.0 0.94C 0.55b 1.7 5.5a medium stress 17.8bC 2.49C 7.1 1.05bC 0.56b 1.9 5.5a severe stress 16.3 e f 2.57bc 6.3 1.00C 0.56b 1.3 5.5a Short Photoperiod control 18.7b 2.70b 6.9 1.25b 0.49b 2.5 5.7a l ight stress 17.6 G d e 2.63bc 6.7 1.09bC 0.54b 2.0 5.5a medium stress 16.l f 2.57bc 6.3 0.95° 0.58b 1.6 5.2a severe stress 16.5 d e f 2.49° 6.6 0.92C 0.55b 1.7 5.1a •Values within a column followed by the same letter are not s ignif icantly different at p = 0. 05. 1 Height to caliper rat io. 2 Shoot to root rat io. 124 significantly s tat i s t ica l interactions occurred between the factors of photoperiod and moisture regime due to the factorial arrangement of the treatments. For every interaction the different response usually occurred at the control moisture stress level between the two types of photoperiod. Significant differences in total height were demonstrated between many of the treatments (Table 4.5). However, with the exception of the control water regime under a natural photoperiod these s tat i s t ical differences probably have l i t t l e operational signficance unless the crop height were approaching the upper or lower limits of the cull standard. Except for the control in a natural daylength, mean heights only ranged from 16.1 cm for the medium stress under a short day to 18.7 cm for the short day control regime, a s t a t i s t i ca l ly significant but operationally small difference of 2.6 cm. There were no apparent trends in total height (Figure 4.5). When moisture stress effects were analyzed under a short day regime, no differences were found between the l i gh t , severe and medium stress levels . The regularly watered plants were s ignif icantly ta l le r by 1.1 to 2.6 cm (Table 4.6). In the natural photoperiod, only the regularly watered plants had s ignif icantly greater total height. When the effects of photoperiod were examined at each moisture regime, significant differences were found at the control level (Table 4.6). Natural photoperiod produced a ta l l e r plant by an average of 4.6 cm. A significant increase of 1.7 cm in a natural photoperiod was shown at the medium moisture stress class. Photoperiod did not s ignif icantly affect caliper growth at any of the moisture stress levels (Table 4.7). However, under a natural photoperiod the control water regime s ignif icantly increased caliper growth (Figure 4.6). No Control Light Medium Severe MOISTURE REGIME Figure 4.5. The final height of Douglas-fir seedlings treated with moisture stress, short days or a combination of both. (Vertical bars indicate standard error of the mean.) 126 Figure 4.6. The effects of moisture stress and photoperiod on the final caliper measurement of Douglas-f i r seedlings in January 1985. (Vertical bars indicate standard error of the mean.) 127 Table 4.6 The effects of photoperiod and moisture regime on total height in Douglas-fir seedlings. MOISTURE STRESS fii2I9fIB152 REGIME Natural Short total height (cm) Control 23 .3 3 * 1 1 8 . 7 a I 1 b l * * hT Light 17.4 D i 17.6D 1 Medium 17.8 b I 1 6 . 1 b H Severe 16.3 b I 16.5 b I •Values within a column followed by the same letter are not s ignif icantly different at p = 0. 05. •Values within a row followed by the same Roman number are not s ignif icantly different at p = 0.05. Table 4.7 The effect of photoperiod and moisture regime on caliper growth of Douglas-fir seedlings. MOISTURE STRESS £M5£i.£!2P REGIME Natural Short caliper (mm) Control 2 .98 3 * 1 2 .70 a I hT** a T Light 2.47D 1 2 .63 3 1 Medium 2.49 b I 2 .57 a I Severe 2.57 b I 2 .49 a I •Values within a column followed by the same letter are not s ignif icantly di fferent at p = 0.05. ••Values within a row followed by the same Roman number are not s ignif icantly di fferent at p = 0. 05. 128 differences were detected between the remaining moisture stress levels . Caliper was unaffected by moisture stress in a short day regime. Shoot dry weight was affected by photoperiod and moisture regime (Figure 4.7). A significant interaction between these two factors also occurred. Under a natural photoperiod, regular watering s ignif icantly increased shoot dry weight compared to the three levels of moisture stress (Table 4.8). There were no differences between a l i gh t , medium or severe moistures stress. Regular watering under a short photoperiod also increased shoot dry weight compared to a severe or medium stress level but not compared to a l ight moisture stress regime. Shoot dry weight was also similar among short day seedlings pretreated with a severe, medium or l ight stress. Photoperiod s ignif icantly affected shoot dry weight but the magnitude and direction of the response varied at each pretreatment moisture regime. At the control and medium stress regimes, shoot dry weight increased signif icantly under a natural photoperiod; although response magnitude was greater in the control treatment. Shoot dry weight was greater in the short day regime at the l ight stress l eve l . No difference between photoperiods occurred at the severe moisture regime. Once again i t can be argued whether these s tat i s t ica l differences are operationally important. In the l ight to severe moisture stress regimes for both photoperiods shoot dry weight only ranged from 0.92 to 1.09 g, a difference of 0.17 g. In an operational perspective i t would seem that only regularly watered plants under a natural daylength had greater shoot dry weights. Under a natural photoperiod, shoot dry weight was 1.74 g and under a short day i t was 1.25 g. When a l l treatment combinations were compared in 129 1.9 0.8 1 1 ! ; 1 Control Light Medium Severe MOISTURE REGIME Figure 4.7. The effects of moisture stress and photoperiod on the final measurement of shoot dry weight of Douglas-fir seedlings in January 1985. (Vertical bars indicate standard error of the mean.) 130 Figure 4.8. The effects of moisture stress and photoperiod on the final root dry weight measurement of Douglas-fir seedlings in January 1985. (Vertical bars indicate standard error of the mean.) 131 Table 4.8 The effect of photoperiod and moisture regime on shoot dry weight accumulation of Douglas-fir seedlings. MOISTURE STRESS PHOTOPERIOD REGIME Natural Short shoot dry weight (g) Control 1.74 a* 1 1 .25 a I 1 Light 0 .94 b I * * 1 . 0 9 a b H Medium 1 . 0 5 b H 0.95 b I Severe 1.00 b I 0.92 b I •Values within a column followed by the same letter are not s ignif icantly different at p = 0.05. •Values within a row followed by the same Roman number are not s ignif icantly different at p = 0.05. Table 4.9 The effects of photoperiod and moisture regime on root dry weight of Douglas-fir seedlings. MOISTURE STRESS REGIME Natural PHOTOPERIOD Short Control Light Medium Severe 0.83 0.55 0.56 0.56 a*I b i b i b i root dry weight (g) 0.49 0.54 0.58 0.55 a l l al al al •Values within a column followed by the same letter are not s ignif icantly different at p = 0.05. ••Values within a row followed by the same Roman number are not s ignif icantly different at p = 0.05. 132 a multiple range test , only the control moisture stress plants under a natural photoperiod were s ignif icantly greater than a l l other treatments (Table 4.5). Although, significant differences were s t i l l shown between various other treatment combinations, i t is doubtful whether i t was of operational significance. Although an analysis of variance indicated that root dry weight was affected by photoperiod and moisture stress pretreatment, these effects were only evident at one particular treatment combination (Table 4.5, 4.9). Regularly watered seedlings under a natural photoperiod had s ignif icantly greater root biomass. Under a short day regime, moisture stress pretreatment did not affect root dry weight. Short days only reduced root dry weight at the control moisture regime. This effect did not occur at any other moisture level (Figure 4.8). The final morphology measurement analyzed was bud height. Neither photoperiod or moisture stress s ignif icantly affected bud height (Table 4.5). 4.3.5 Root Growth Capacity (RGC) Lif t RGC and post storage RGC values were very similar (Table 4.10). Since these values from both sample periods were s imilar , treatment effects wil l only be discussed for one data set, January l i f t RGC. The effects of one factor were analyzed by each level of the second factor because of a significant interaction between photoperiod and moisture stress. Pretreatment moisture regime significantly affected RGC but photoperiod did not. Under a natural daylength, RGC generally declined with increasing 133 Table 4.10 The effect of moisture stress on root growth capacity of Douglas-fir seedlings under two photoperiods. MOISTURE STRESS ROOT GROWTH CAPACITY UNDER TWO PHOTOPERIODS REGIME Natural Short 1. January Lift control 4.00 a* 2.9 & light stress 2 . 5 0 ° 2.45 a medium stress 2 . 9 0 ° 2.95 a severe stress 1.65c 2.75 a 2. Post Storage control 3.95 a 2.60 3 l ight stress 2 . 4 5 ° 2.85 a medium stress 2 . 6 5 ° 2.65 a severe stress 1.65c 2.85 a •Values within a column of a numbered section are not s ignif icantly different at p = 0.05. 134 4.5 Figure 4.9. The effects of moisture stress and photoperiod on root growth capacity of Douglas-fir seedlings measured in late January 1985. (Vertical bars indicate standard error of the mean.) 135 moisture stress (Figure 4.9). The control RGC was 4.0 which was s ignif icantly greater than the other moisture stress levels (Table 4.10). The RGC values for the l ight and medium levels were similar at 2.50 and 2.90, respectively. RGC was s ignif icantly lower in the severe stress with a value of 1.65. However, the effect of pretreatment moisture stress was not evident under a short day regime where no differences in RGC were demonstrated between any of the moisture stress levels . 4.3.6 Dormancy Intensity Days to bud break or dormancy intensity at the January l i f t s ignificantly decreased with short day treatment at the control and medium stress moisture regimes (Table 4.11). Short photoperiod reduced days to bud break at the l ight and severe moisture stress levels but not s igni f icant ly . Therefore, bud act iv i ty generally increased in seedlings pretreated with short days (Figures 4.10, 4.11). In a short photoperiod, moisture stress pretreatment did not affect dormancy intensity. Under a natural photoperiod, s ignif icantly more days were required to break buds of the regular watered seedlings. No differences were demonstrated between the l i ght , medium and severe moisture stress levels . Dormancy intensity after five weeks of cold storage decreased signif icantly under a short photoperiod, irrespective of moisture stress (Table 4.11). Moisture stress had no significant effect on dormancy intensity under either photoperiod. 136 Table 4.11 The effect of photoperiod and moisture stress on dormancy intensity of Douglas-fir seedlings. Tests were conducted during the January l i f t and after five weeks of cold storage. MOISTURE STRESS REGIME DORMANCY INTENSITY UNDER TWO PHOTOPERIODS Natural (days to bud break) Short (days to bud break) 1. January Li f t control l ight stress medium stess severe stress 21.4 18.8 18.7 17.2 a* I bl*< bl bi 15.8 17.3 17.2 16.4 a l l al a l l al 2. Post Storage control l ight stress medium stress severe stress 23.1 21.8 22.7 24.9 al al al al 14.5 17.7 18.3 14.4 a l l a l l a l l a l l •Values within a column of a numbered section followed by the same letter are not s ignif icantly different at p = 0.05. ••Values within a row of a numbered section followed by the same Roman number are not s ignif icantly different at p = 0.05. 137 26 Control Light Medium Severe MOISTURE REGIME Figure 4.10. T h e effects of moisture stress and photoperiod pretreatment on dormancy intensity of Douglas-f i r seedlings in January 1985. (Vertical bars indicate standard error of the mean.) 138 26 24 w T3 22-ro 20 >-t CO UJ r -Z >-o z < cc o D 18 16 14 12 / / / \ \ \ \ Legend A Noturol Photoperiod X Short Days Control Light Medium MOISTURE REGIME Severe Figure 4.11. The effects of moisture stress and photoperiod pretreatment on dormancy intensity of Douglas-f i r seedlings in March 1985. (Vertical bars indicate standard error of the mean.) 139 No s tat i s t ica l comparisons between the two sample periods were made since the test environments were dissimilar. The January test was done in a 20°C thermoregime while the March test was conducted under a daily temperature of 30°C and a nightly temperature of 25 °C . However, inspite of the warmer temperatures of the March test, more days were required to induce budflush in seedlings under a natural daylength. 4.3.7 Frost Hardiness At the January l i f t no differences in frost hardiness were demonstrated between any of the treatment combinations. At a test temperature of - 2 1 ° C , only 0 to 2.5% of the sample seedlings died throughout a l l treatments. However, greater than 50% of a l l test seedlings in a l l treatment combinations were dead at - 2 4 ° C . Hence, the LT5 Q for a l l treatments was between -21°C and - 2 4 ° C . Neither moisture stress or photoperiod affected frost hardiness five months after the treatments were applied. 4.4 Discussion The effects of moisture stress, short days and a combination of both reported in this study must be interpreted with caution for several reasons. F i r s t , the assumption of homogeneous variance was frequently not met. This must be considered when treatment means were demonstrated by a multiple range test to be s ignif icant. Secondly, the effects of treatment combinations on such variables as budset incidence, morphology and root growth capacity may partly reflect the influences of operational practices applied prior to project i n i t i a t i o n . A specific example of this is the budset incidence recorded after only 16 days of treatment. Even 44% of the regularly watered 140 seedlings in a natural photoperiod had formed terminal buds. Two weeks later 84% of al l these seedlings had flushed. An examination of the operational pretreatment irr igat ion regime accounts for this occurence. At the date of the project in i ta t ion , nursery staff had already commenced an overall moisture stress regime to slowly reduce height growth throughout the f i r crop. By mid-July the average stressed 313 block weight in greenhouse s ix , the location of the research t r i a l , was only 4.3 kg. This corresponded to a medium moisture stress in this research project. Since the experiment population was selected from the operational stock in July, a l l seedlings had received a moisture stress regime prior to project in i t i a t i on . For this reason, the fast rate of bud formation was most l ike ly affected by the pretreatment operational i rr igat ion regime. The high rate of reflushing evident in the control seedlings under a natural daylength occurred because once the study began frequent irr igat ion maintained control blocks around 5.5 kg. The operational i rr igat ion regime may also explain the diss imilar i ty in the RGC and root dry weight results between Study I and Study II. In Study I four weeks of short days in a frequent irr igation regime did not reduce RGC or root biomass in Douglas-fir seedlings compared to that in plants under a natural daylength. Short days reduced these characteristics in Study II. The operational i rr igat ion regime applied in late June and early July 1984 may have affected these results. In 1983 seedlings were frequently watered prior to and during the dormancy induction project. A diss imilari ty in thermoregime also existed between the short day and natural daylength treatments. The greenhouse where short days were applied was at least 5°C warmer during the day. Temperatures frequently exceeded 141 35°C . This high thermoregime may also have affected seedling vigour and root growth response. Interpretation of the data was further complicated by the significant interactions between moisture regime pretreatment and photoperiod. In the morphology data, the interaction was usually between the two photoperiods at the control moisture regime. Reasons for this occurrence are obvious. An eight hour day, regardless of moisture regime, and the selected levels of l i ght , medium and severe moisture stress under a natural daylength are environmental conditions which promote dormancy induction and inhibit shoot growth of coastal Douglas-fir seedlings. Frequent irr igat ion that v i r tua l ly eliminates plant moisture stress encourages growth in the growing season. When this was combined with a late July and early August photoperiod, conditions remained favourable for shoot elongation. Hence this treatment combination was the only one which promoted shoot growth for a longer period of time. The interaction between photoperiod and plant moisture stress was shown as significant because at the same control moisture l eve l , growth was inhibited by the inductive short day regime. For a l l other moisture stress levels , growth stopped regardless of photoperiod regime. The effect of moisture stress or short days on reduced height growth, root dry weight and shoot dry weight coincides with results documented in the l i terature . Dry weight accumulation in the shoots of black spruce seedlings and height increment decreased signif icantly when photoperiod was reduced from 15 to 8 hours (D'Aoust and Cameron 1981). Daylength had less of an effect on root dry weight accumulation. The fresh weight of Douglas f i r seedlings declined under a nine hour day (Lavender and Wareing 1972). When 142 container Douglas-fir seedlings were subjected to moisture stress of -6 and -12 bars in the 8th through the 16th week from sowing and the 12th through the 21st week, the higher stressed seedlings had lower shoot dry weights of 32% and 38% for the two treatment dates and lower root dry weights of 12% and 35% (Timmis and Tanaka 1976). The results of this thesis study are similar to those of Timmis and Tanaka (1976), where root dry weight declined from 0.83 g at a stress treatment of -4.9 bars to 0.55 g at a stress of -9.5 bar, a decrease of 34%. However, stresses of -17.5 and -23.4 bars resulted in no further decrease in root biomass. A similar trend was evident for shoot dry weight. When Blake et a l . (1979) applied plant moisture stresses of -6 to -8 bars to bareroot Douglas-fir seedlings, the effect of the stress on root dry weight depended upon the date of treatment i n i t i a t i o n . Plants stressed on July 15 had s ignif icantly larger root dry weights than seedlings treated later in the summer. Only the plants which received treatment in September had s ignif icantly reduced root dry weight. Cheung (1973) concurrently applied moisture stress or short days to western hemlock container seedling. Total height, shoot dry weight and root dry weight s ignif icantly decined under either treatment. Shoot dry weight was reduced to 0.359 g in a short day regime and to 0.490 g with moisture stress when treatments were ini t iated 16 weeks from sowing. Shoot dry weight was 0.69 0 g in seedlings under a natural daylength. Root dry weight declined in both treatments compared to the control but the difference was not s ignif icant. The effect of the dormancy induction treatments of this project on shoot and root dry weight are in agreement with the above reported studies. 143 That i s , short days, a l ight to severe moisture stress, or a combination of both reduced shoot and root dry weight compared to a control moisture regime under a natural photoperiod. However, the short day effect on shoot dry weight accumulations diminished under increased levels of moisture stress. Short photoperiod did not influence root biomass in a l ight to severe stress. The influence of short days on root dry weight apparently coincides with those of Cheung (1973) and Timmis and Tanaka (1976). A l ight to medium stress under a natural daylength reduced caliper growth compared to the unstressed treatment. This result is in agreement with Timmis and Tanaka (1976) who demonstrated that caliper growth was reduced by 21% and 30% in seedings stressed to -12 bars at two in i t i a t ion dates compared to plants stressed to -6 bars. Blake et a l . (1979), however, showed that Douglas-fir caliper growth was unaffected by time of application and a stress treatment of -6 to -8 bars. Under a short day regime, moisture stress did not affect caliper growth in this thesis project. The final morphological characteristic analyzed was bud height. No treatment effects were demonstrated. This result seems surprising in view of the findings reported by Colombo and Smith (1984) who reported that delayed budset in i t i a t ion reduced the number of needle primordia in black spruce container seedlings. However, the delay in bud formation occurred in September and October when cooler fa l l temperatures probably affected bud maturation. The delay in bud formation in the control seedlings under a natural daylength occurred in the summer. By September 3, 76% of the terminal buds had formed. Bud maturation proceeded under warm fa l l temperatures. The delay in budset in i t ia t ion was probably insufficient to influence bud height. In addition to time of bud formation, seedling vigour 144 at the time of dormancy induction is also important to the resultant bud size and the number of primordia within the bud (Thompson 1985). Bud height is also an indication of potential shoot growth in the f i e l d . If this is so, potential f ie ld performance of the seedlings in this study was possibly unaffected by the various induction treatments. However, i t would seem unwise to infer such a conclusion from one test. Of the three physiological attributes measured, only frost hardiness was unaffected by moisture stress or short day treatments. It is unfortunate a hardiness test was not conducted shortly after treatment completion. Evidence in the l i terature demonstrates that frost hardiness differences occur when Douglas-fir seedlings are treated with short days, long days or moisture stress. When bareroot Douglas-fir seedlings were treated with eight weeks of 8 or 10 hour days, frost hardiness was enhanced not immediately upon treatment completion, but two and four weeks after treatment (McCreary et a l . 1978). That i s , the ab i l i ty to acclimate quickly improved after short day pretreatment. Tanaka (1974) reported a similar result for container Douglas-fir seedlings. Several other studies concur with these findings (Aronsson 1975; Christersson 1978; D'Aoust and Cameron 1981; McGuire and Fl int 1962; Rosvall-Ahnebrink 1981; van den Driessche 1970). Evidence on the influence of moisture stress on cold hardiness in coniferous species of the Pacific Northwest is confl ict ing. In a controlled moisture stress of -6 and -12 bars applied in the 8th through 16th weeks from sowing and the 12th through 21st weeks, Douglas-fir container seedlings were tested for frost hardiness immediately after treatment, after 5.5 weeks of cold treatment at 5°C in an eight hour photoperiod, and after 11.5 weeks of similar cold treatment. Hardiness was similar between both stress levels 145 immediately after treatment. After eleven weeks of cold treatment, seedlings pretreated with the milder stress were 4 to 5°C hardier than the higher stressed plants. Blake et a l . (1979) appied three levels of moisture stress, 0 to -5, -5 to -10, and -10 to -15 bars, to bareroot Douglas-fir seedlings in late July. A mild stress of -5 to -10 bars enhanced hardiness, measured in October and December, to that of the control while the higher stress level reduced i t . When the seedlings of this study were l i f t ed in January, no differences in hardiness were detected between moisture stress or short day treatments. The findings of van den Driessche (1969b) provide an explanation for this occurrence. After six weeks, an eight hour photoperiod s ignif icantly increased frost hardiness levels in Douglas-fir compared to a twelve or sixteen hour daylength. Moisture stress did not affect frost hardiness but photoperiod increased hardiness more under a well watered regime compared to increasing moisture stress. When non-hardy plants were grown in the natural short days of autumn or under an extended photoperiod, short day seedings quickly acclimated after mid-October. The long day plants had delayed hardiness until mid- November. By mid-December hardiness levels were similar in both groups of seedlings. These findings possibly explain why no differences were detected in frost hardiness between seedlings treated with short days, long days or moisture stress. Differences in hardiness probably occurred after the treatments were completed and persisted throughout the early f a l l . However, under the naturally declining short days and decreasing temperatures of late autumn and early winter, seedlings from al l treatments acclimated to similar levels , irrespective of any probable early fa l l d i s s imi lar i t ies . 146 The RGC results indicate that seedling vigour was similar among seedlings from many of the treatment combinations. Varying levels of moisture stress did not affect RGC under a short day regime. That i s , RGC was similar in a l l the short day treatments. Although an analysis of variance indicated that photoperiod did not influence RGC, a multiple range test on a l l treatment combinations indicated that under a control moisture regime, short days s ignif icantly reduced RGC compared to a natural photoperiod. This result is in agreement with Lavender and Wareing (1972) who showed that root act iv i ty declined in seedlings pretreated with short days. It must be emphasized that the short day effect was not evident under the other moisture regimes. Under a natural daylength, RGC declined with increasing moisture stress. Increased moisture stress somehow affected root growth and the ab i l i ty to regenerate new roots. Root growth and its regenerative capacity may be influenced by moisture stress through its effect on the foliage. F i r s t l y , photosynthetic capacity decreases with plant water deficits (Kramer and Kozlowski 1979). Under repeated drying cycles photosynthesis does not always resume to predrought levels when rewatered. Thus, one possible explanation for reduced root growth is a reduction in the production of current photosynthates. However, i f this occurred, root dry weight should have further declined under the short day regime because of the reduction in hours available for photosynthesis. Yet, no significant differences were shown between a l ight to severe moisture stress for either photoperiod. Under a control moisture regime, short days significantly reduced root growth and root growth capacity. Lavender and Hermann (197 0) reported that the production of substances such as hormones or sugars from mature foliage 147 were necessary for active root growth. This, in turn, was influenced by daylength (Lavender and Wareing 1972). Since daylength influences this process of exporting growth regulating hormones, i t is interesting to note that RGC and root growth, on a dry weight basis, was similar among a l l short day treatments, regardless of moisture stress, and the l ight and medium stresses under a natural photoperod. Possibly both these environmental signals influence the export of growth regulating hormones in a similar manner. The main factor common to a l l these treatments is that dormancy was init iated at the same time. Hence, the shift in balance of growth promoting and inhibiting hormones occurred at similar times. A final explanation for the differences in RGC and root biomass is that the preexperimental i rr igat ion regime combined with the induction treatments of moisture stress and short days, with its high temperature regime, reduced overall plant vigour. This was consequently expressed in the root growth capacity results. In the dormancy intensity tests, short days accelerated bud break compared to a natural photoperiod. This result is in agreement with Lavender and Wareing (1972) who demonstrated that bud act ivity increased in seedlings pretreated with short days. Accelerated budflush was reported in other studies (Sandvik 1980; Rosvall-Ahnebrink 1981). A theory for this phenomenon is that photoperiod affects the growth potential of Douglas-fir through its influence on growth regulating hormones within the bud (Lavender and Hermann 1970; Lavender and Wareing 1972). The dormancy intensity results under a natural daylength suggest storage was a physiological stress to these seedlings. Budflush is predominantly a temperature mediated response as ch i l l ing require- ment becomes f u l f i l l e d (Campbell 1978; van den Driessche 148 1975). A faster rate of budflush should have occurred in the warmer March post storage test compared to the January l i f t test. Instead, days to budburst increased after the storage period. The selection of the optimum treatment for controlling height growth while maintaining quality in a Douglas-fir container crop is not possible from this study. The control moisture stress regime under a natural photoperiod had the best morphological characteristics with respect to caliper and root biomass, the highest RGC and comparable frost hardiness at the time of the January l i f t . This regime does not permit the manipulation of height growth. The cessation of shoot growth is controlled by the natural photoperiod. The timing of dormancy induction is not necessarily controlled. In evaluating the remaining treatment combinations, short photoperiod with frequent irr igat ion possibly yielded a s l ightly better qualty seedling. This conclusion is based on the s l ightly higher shoot dry weight and cal iper , comparable root growth capacity and frost hardiness, and rapid and homogeneous budset incidence with a low incidence of reflushing. Accelerated spring budflush is another possible advantage. However, a final conclusion about this regime should not be made from this study because of the pretreatment operatonal i rr igat ion regime and the high thermoregime during experimentation. In view of the different findings between Study I and Study II, i f proper materials which reflect radiation and ventilate the system can not be supplied, short days have l i t t l e benefit but quick control of height growth and accelerated spring budflush. When the improved height growth performance of outplanted seedlings in Study I is considered, this benefit may prove worthwhile especially on sites with excessive brush competition or 149 mid-summer water def ic i t s . At present, there are insufficient studies, and growth and yield tables which demonstrate the importance of rapid and increased growth performance in the f i r s t few years of a new plantation. Therefore, the benefit of increased f i r s t year height growth cannot be truly evaluated in this thesis. The results from this study suggest that there is l i t t l e benefit in applying short days to seedlings already treated with three possible levels of moisture stress for 16 days. The only possible advantage is to minimize the incidence of reflushing. However, once moisture stressed seedlings set buds, maintenance of a proper moisture regime should also prevent a second flush. The evidence from the morphology, frost hardiness and root growth capacity data suggest that the l ight and medium moisture stress regimes also effectively init iated budset after a six week period and maintained seedling quality compared to the short day regimes. However, a severe moisture stress is not recommended because of i ts effect on root growth capacity. 4.5 Conclusions Short days effectively init iated and maintained budset in Douglas-fir seedlings in four weeks. After six weeks, a l ight to severe moisture stress was as effective as short days in controlling height growth. Unstressed seedlings in a natural photoperiod had the slowest rate of bud formation and the highest incidence of llamas growth. Severe, medium, l ight and control moisture stress treatments corresponded to average predawn shoot water potential measurements of -23.4, 150 -17.5, -9.5 and -4.9 bars respectively. The operational method of weighing styroblock weights as an indication of moisture stress was related to plant moisture stress, but once 313 block weights approached 4.5 kg, small reductions in block weight produced major increases in plant moisture stress. Short days, irrespective of moisture stress, and a l ight to severe moisture stress under a natural photoperiod signif icantly reduced total height and caliper compared to the control moisture regime in a natural photoperiod. With the exception of the l ight stress l eve l , short days reduced shoot dry weight accumulations. In addition, control or regularly watered seedlings had greater shoot dry weights than l ight to severe moisture stresses. Short days and a l ight to severe moisture stress s ignif icantly reduced root dry weight compared to a control, natural photoperiod treatment. Among these treatment combinations, however, no significant differences occurred. In a comparison of a l l treatment combinations, only the control plants under a natural photoperiod were significantly larger in a l l morphological properties than seedlings from al l other treatments. Although significant differences were s t i l l shown between these other treatments, i t seems doubtful whether these differences were operationally s ignif icant. That i s , short days, moisture stress or a combination of both had similar effects on reducing the seedling morphological characteristics of height, cal iper , shoot dry weight and root dry weight. Unstressed seedlings in a natural daylength had the highest value of root growth capacity. All other short day and moisture stress treatments 151 reduced root growth capacity. Most treatments had similar levels . Only the severe stress under a natural photoperiod reduced root growth capacity below any other treatment combination. Five weeks of cold storage had no effect on root growth capacity. Short days, irrespective of moisture stress, accelerated bud burst in January and March dormancy intensity tests. Moisture stress had no effect on bud act iv i ty . Five months after the treatment period, no differences in frost hardiness were detected between any of the treatment combinations. All seedlings were hardy to at least - 2 1 ° C . In spite of any differences in hardiness levels that moisture stress or short days may have i n i t i a l l y produced in the early f a l l , neither of these factors influenced frost hardiness after five months of autumn and winter temperatures and photoperiods. The physiological and morphological characteristics of a l l seedlings may partly reflect the influence of the operational i r r igat ion regime applied prior to project in i t i a t i on . In late June and early July, seedlings were operationally stressed to a 313 styroblock weight of 4.3kg. This corresponded to a medium moisture stress of -9.5 bars in this research t r i a l . The high thermoregime of the short day greenhouse was another factor which possibly influenced the results. Temperatures which frequently exceeded 35°C may have reduced seedling vigour and growth. The control moisture stress regime under a natural photoperiod had the best morphological characteristics especially root dry weight and cal iper , the highest value of root growth capacity and comparable frost hardiness at the time of the January l i f t . It was the least effective in 152 controlling height growth through early budset in i t i a t i on . The overall thesis objective was to develop a regime which effectively controlled height growth while maintaining seedling quality. Of the remaining treatments, short days with frequent irr igat ion to minimize moisture stress most closely met this objective. Short days produced rapid and homogeneous budset in the Douglas-fir seedlings. The incidence of a second flush was only 4%, the lowest of a l l treatment combinations. 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Cold hardiness testing of container seedlings. Pages 21-25 in Proc. 1981 Intermountain Nurserymen's Assoc. Meeting. Nor. For. Res. Cen. CFS., NOR-X-241. 95. Wareing, P.F. 1956. Photoperiodism in woody plants. Ann. Rev. Plant Physiol. 7:191-214. 96. Wareing, P.F. and P.F. Saunders. 1971. Hormones and dormancy. Ann. Rev. Plant Physiol. 22:261-288. 97. Warrington, J. and D.A. Rook. 1980. Evaluation of techniques used in determining frost tolerance of forest planting stock. A review. N.Z . J . For. Sc i . 10(1) -.116-132. 98. Weiser, C. J . 197 0. Cold resistance and injury in woody plants. Science 169:1269-1278. 99. Worrall, J. and F. Mergen. 1967. Environmental and genetic control of dormancy in Picea abi.es. Phys. Plant 20:733-745. 100. Young, E. and J.W. Hanover. 1978. Effects of temperature, nutrient an moisture stress on dormancy of blue spruce seedlings under continuous l i ght . For .Sci . 24(4): 458-467. 101. Zaerr, J.B. 1985. The role of biochemical measurements in evaluating vigor. Pages 137-141 in Evaluating seedling quality: pr inciples , procedures and predictive ab i l i t i e s of major tests (Duryea, M.L . , ed.) . Forest Research Laboratory, Oregon State University, Corval l i s . 102. Zaerr, J .B . , B.D. Cleary, and J .L. Jenkinson. 1981. Scheduling irr igat ion to induce seedling dormancy. Nurseryman's Assoc. and West. For. Nurs. Assoc combined meeting, Aug. 12 -14, 1980. Boise, Idaho. USDA For. Serv., Gen. Tech.Rep., Int. For. and Range Exp. S t . , No. INT-109., p.71-79. APPENDIX I 162 MONITORING THE ROOT GROWTH CAPACITY OF PLANTING STOCK 1 1. Methods of Measurement Root growth capacity (RGC) tests for assessing stock quality are conducted under standard conditions in the assumption that the results obtained are indicative of relative capacity for root growth under f ie ld conditions. The just i f icat ion for this assumption is the close correlation usually observed between the RGC of stock measured under highly favorable conditions in the laboratory and its performance (early survival X growth) in the f i e l d . 1.1 Test Conditions To obtain results quickly, RGC tests are usually conducted under conditions thought most favorable to root growth. Although the optimum conditions have not been precisely determined, rapid root growth has been observed in interior spruce, lodgepole pine, interior and coastal Douglas f i r , western hemlock and western red cedar under the following conditions: 1 Reproduced from N. Burdet. 1983. Controlling the root growth capacity planting stock. M.O.F. Unpublished report. 163 Standard RGC Test Conditions day temperature 3CPC. night temperature 2 5 ° C . daylength 16 hr. l ight intensity 400 tiErn^S" 1 relative humidity 75% 1.2 Root Environment Test seedlings may be planted in a defined solid medium or grown either hydroponically or with their roots enclosed in a mist chamber. Adequate root aeration is d i f f i cu l t to achieve in a hydroponic system. Good results have been reported with the root mist chamber which is to be used for root growth testing in Ontario's nurseries. The reason cited for preferring this system over the use of a solid medium is the saving in labour in planting trees and then washing the roots for examination at the end of the test. It has also been suggested that root growth is more rapid in a mist chamber than in a solid medium. Nevertheless, until local experience has been gained with the root mist chamber, i t seems advisable that operational RGC testing in B.C. be conducted with stock planted in a standard solid medium. A suitable medium is a 3:1 mix of peat and vermiculite (adjusted to pH 4.5 to 5.0 with dolomitic lime). 1.3 Measurement Root growth can be measured volumetrically, by surface area, or by the number or length of new roots. Measurement by volume or area increase is quantitative but subject to error due to decay and loss of 164 old roots during the test . Measuring the length of new roots formed is a more reliable quantitative technique, but i t is extremely time-consuming and therefore, unacceptably expensive for use in routine monitoring of stock quality. Counting new roots more than a certain length, (usually 1 cm) is a less time-consuming method of estimating root growth, although i t is not precisely quantitative. Root counting can be greatly speeded up by recording root numbers in broad classes such as the following: Index of root growth (IRG) New Roots 0 None 1 Some, none > 1 cm in length 2 1-3 > 1 cm 3 4-10 > 1 cm 4 11-30 > 1 cm 5 31-100 > 1 cm 6 101-399 > 1 cm 7 more than 3 00 > 1 cm Using this scale i t is frequently unnecessary to count more than a minority of the roots since, when one class boundary is reached, e .g . , 3, 10, 30 or 100, i t is often clear that the next boundary wil l not be exceeded. The precision of this method of estimating root growth is not great. The range of variation in the RGC of forest nursery stock i s , however, enormous so that a crude scale is quite adequate as a basis for 165 segregating stock into a number of RGC grades. For purposes of quality control, this is a l l that is required since only major differences in RGFC have appreciable effects on f ie ld performance. 1.4 Duration of Tests Under the standard test conditions noted above, a mean IRG of more than 5 after 1 week has been observed in batches of a l l species tested (Table I). Thus a one week test is long enough to divide stock into a number of RGC grades. 166 APPENDIX II THE WHOLE SEEDLING ASSESSMENT METHOD FOR FROST HARDINESS EVALUATION BY C. J . SALLY JOHNSON, SEEDLING QUALITY SERVICES One hundred seedlings were randomly selected from each treatment and shipped to Seedling Quality Services. Four subsamples of 20 seedlings respectively received four separate freezing temperatures of -21.0; -24.1, -25.2 and - 2 6 . 1 ° C . Twenty seedlings were maintained as controls. A l l test seedlings were placed in a favourable environment and were subsequently assessed for frost injury to needles, buds and the stem cambium. Damage to each of these tissues was rated as follows: I DAMAGE SCALE 1. Needles 0 - 1 0 with 0-0% dead needles 10-100% dead needles 2. Terminal Buds 0 - 1 with 0 = 1 ive bud 1 = dead bud 3. Lateral Buds 0 - 9 with 0=0% dead buds 9 = 100% dead lateral buds 167 4. Stem 0 - 4 with 0 = no stem dead 1 = top 1/4 stem dead 2 = top 1/2 stem dead 3 = entire stem dead 4 = stem girdled in lower 1/4 and therefore dead II DAMAGE SCALE Viabi^i_ty_rating _ Needles Buds Stem 0 (economically alive) 0 - 10 0 - 8 0 - 1 0.5 (half k i l l ) 0 - 10 0 - 5 0 - 2 0 - 10 8 0 - 1 1.0 (k i l l ) 0 - 10 8 - 9 2 - 4 0 - 10 0 - 7 3 - 4 0 - 1 0 9 0 - 2 From the damage scale assigned to the different tissues, a v i ab i l i ty rating is assigned in order to determine the LT 5 0 value. If one of the l i s t temperatures is not this value, i t is extrapolated from a graph where damage is plotted against temperature. APPENDIX I l ia CROP HISTORY OF DOUGLAS-FIR SEEDLINGS FROM STUDY I 169 CROP HISTORY RECORD SEEDLOT: 4390 SPECIES: Fc CONTAINER SIZE: 40 c u . i n . CONTAINER TYPE: SB313 DATE SOWN: Mar. 23, 1984 GREENHOUSE HISTORY WEEK FERTILIZER PESTICIDE TYPE/AMOUNT TYPE/AMOUNT WATER April 17 7-40-17/100L-14.3kg 23 7-40-27/100L-14.3kg 25 7-40-27/100L-14.3kg 29 7-40-27/100L-14.3kg May 5 Watered only 10 Watered only 15 Watered only 21 Watered only 28 Bravo/Safers Soap June 9 Soilwet/lOOc 15 Watered only 21 Bravp/Safers Soap Watered only 28 Watered only July 11 Soilwet/lOOc 19 4-25-35/1 001-10kg 28 Watered only 30 Bravo August 5 4-25-35/100L-1Okg 12 4-25-35/1001-10kg 2 0 4-25-35/1 00L-1 Okg 31 4-25-35/100L-1 Okg Sept. 1 Bravo 23 4-25-35/100L-1Okg 27 Bravo/Safers Soap Nov. 7 Bravo 28 Ca(N03)2/15.5kg Bravo APPENDIX 11 lb CROP HISTORY OF DOUGLAS-FIR SEEDLINGS FROM STUDY II 171 CROP HISTORY RECORD SEEDLOT: 4371 SPECIES: Fc CONTAINER SIZE: 4 cu . in . CONTAINER TYPE: SB313 DATE SOWN: Mar. 3 0, 1983 GREENHOUSE HISTORY WEEK FERTILIZER PESTICIDE QUANTITY OF TYPE/AMOUNT TYPE/AMOUNT WATER 1. l-52-17/33L-8.8Kg Kept Moist 2. /40L 3. 2O-2O-2 0/37L-12.5Kg 4. 2 0-2 0-2 0/37L 5. 2O-2O-20/24L 1 L 6. 2 0-2 0-2 0/72L-15kg 1/2 hr 7. 20-20-20/31L 8. 20-20-20/75L 9. 20-20-20/37L 10. 2 0-2 0-2 0/88L 11. 20-20-20/91L 12. 20-2 0-2 0/66L 80n/Bravo/275L 13. 2O-2O-20/56L 3/4 hr 14. 20-20-20/76L 15. 8Qn/Bravo/275L Flushed out 16. /115L 17. 2O-2O-2 0/42L-12.5kg 18. 2 0-2 0-2 0/84L 19. 2 0-2 0-2 0/135L 8Qn/Bravo/275L 2 0. 2 0-2 0-2 0/89L 8Qn/Bravo/275L APPENDIX IV HOMOGENEOUS VARIANCE CHECK OF DATA ANALYSES STUDY TABLE VARIABLE HOMOGENEOUS VARIANCE I 3.2 height caliper * shoot dry weight root dry weight 3.3 height * caliper * shoot dry weight * root dry weight * 3.4 height * caliper * shoot dry weight * root dry weight * 3.5 height * caliper * shoot dry weight * root dry weight * 3.6 caliper * 3.7 shoot dry weight * 3.8 shoot dry weight 3.11 RGC * 3.15 survival 3.16 survival 3.17 total height * caliper * height increment * relative growth rate * 3.18 total height caliper height increment * relative growth rate APPENDIX IV HOMOGENEOUS VARIANCE CHECK OF DATA ANALYSES STUDY TABLE VARIABLE HOMOGENEOUS VARIANCE II 4.2 bud formation * 4.3 bud formation 4.4 4 week bud formation 4 week bud flushing 6 week bud formation 6 week flushing 4.5 height cali per shoot dry weight * root dry weight bud height 4.10 January RGC * March RGC * 4.11 January dormancy intensity * March dormancy intensity * 174 APPENDIX V ANALYSIS OF VARIANCE TABLES A. CHAPTER THREE ANOVA TABLES 1. ANOVA FOR TABLE 3.2: August Morphology Measurement on Hemlock, i . Height. ANOVA Source df ss ms F-ratio Probability Photoregime 4 977. 04 244.26 27.99 0. 0000 Error 95 828.99 8.73 Total 99 186.0 i i . Caliper. ANOVA Source df ss ms F-ratio Probability Photoregime 4 1.6 9 0.42 3 . 09 0.019 Error 95 12.99 0.14 Total 99 14.67 175 i i i . Shoot Dry weight. ANOVA Source df ss ms F-ratio Probability Photoregime 4 0.95 0.24 6.07 0.0005 Error 45 1.76 0.039 Total 49 2.71 iv . Root dry Weight ANOVA Source df ss ms F-ratio Probability Photoregime 4 0. 083 0.0021 3.40 0. 0164 Error 45 0.28 0. 0061 Total 49 0.36 176 2. ANOVA FOR TABLE 3.3: August Morphology Measurements in Douglas-fir. i . Height. ANOVA Source df ss ms F-ratio Probability Photoregime 4 476.35 119.09 22.53 0. 0000 Error 45 237.83 5.29 Total 49 714.19 i i . Caliper ANOVA Source df ss ms F-ratio Probability Photoregime 4 0.46 0.12 1.31 0.2812 Error 45 3 . 9 9 0. 089 Total 49 4.45 177 i i i . Shoot Dry Wei ght ANOVA Source df ss ms F-ratio Probability Photoregime 4 1.12 0.28 3.21 0. 0210 Error 45 3 . 9 0 0. 087 Total 49 5.02 i v . Root Dry Weight. ANOVA Source df ss ms F-ratio Probability Photoregime 4 0. 03 4 0. 008 6 0.9 5 0.4 449 Error 45 0.41 0. 0091 Total 49 0.44 178 3. ANOVA FOR TABLE 3.4: September Morphology Measurements in Hemlock, i . Height ANOVA Source d ss ms F-ratio Probability Photoregime 4 937.94 234.48 22.75 0. 0000 Conditioning 1 3.35 3.35 0.32 0.57 00 Photoregime *Conditioning 4 102.85 25.71 2.49 0. 0484 Error 90 927.65 10.31 Total 99 1971.8 i i . Caliper. ANOVA Source df ss ms F-ratio Probability Photoregime 4 2.75 0.68 3.82 0.0065 Conditioning 1 0.63 0.63 3.51 0.0642 Photoregime *Conditioning 4 0.96 0.24 1.33 0.2627 Error 90 16.19 0.18 Total 99 2 0.53 179 i i i . Shoot Dry Weight ANOVA Source df ss ms F-ratio Probabili Photoregime 4 2.38 0.6 0 5.51 0.0005 Conditioni ng 1 0.89 0.90 8.27 0. 005 0 Photoregime * Conditioning 4 0.32 0.08 0 0.7 4 0.5690 Error 90 9.71 0.11 Total 99 13.31 iv . Root Dry weight. ANOVA Source df ss ms F-ratio Probability Photoregime 4 0.52 0.13 3.12 0. 0187 Conditioning 1 0.29 0.29 6.84 0. 0104 Photoregime *Conditioning 4 0.29 0.074 1.76 0.1437 Error 90 3.76 0.042 Total 99 4.87 180 4. ANOVA FOR TABLE 3.5: September Morphology Measurements in Douglas-fir. i . Height. ANOVA v Source df ss ms F-ratio Probability Photoregime 4 1435.4 358.86 87 .13 0. 0000 Conditioning 1 0.22 0.22 0. 054 0.8174 Photoregime *Conditioning 4 59.83 14.96 3. 63 0. 0086 Error 90 37 0.69 4.12 Total 99 1866.2 i i . Caliper ANOVA Source df ss ms F-ratio Probabi1 Photoregime 4 1.57 0.39 1.62 0.1757 Conditioning 1 1.13 1.13 4.68 0. 03 3 2 Photoregime *Conditioning 4 1.41 0.35 1.46 0.2215 Error 90 21.82 0.24 Total 99 25.94 181 i i i . Shoot Dry Weight. ANOVA Source df ss ms F-ratio Probability Photoregime 4 11.16 2.79 15.74 0. 0000 Conditioning 1 2.12 2.12 11.95 0. 0008 Photoregime *Conditioning 4 1.19 0.3 0 1.68 0.1619 Error 90 15.95 0.18 Total 99 30.41 iv . Root Dry Weight. ANOVA Source df ss ms F-ratio Probabil Photoregime 4 0.24 0.059 1.56 0.19 05 Conditioning 1 0.38 0.38 10.08 0. 0021 Photoregime *Conditioning 4 0.17 0. 044 1.16 0.3354 Error 90 3.39 0.038 Total 99 4.18 182 5. ANOVA TABLES FOR TABLE 3.11: RGC. i . Douglas-fir. ANOVA Source df ss ms F-ratio Probability Photoregime 4 24.00 6.00 19.29 0. 0000 Error 45 14.00 0.311 Total 49 38. 00 i i . Western Hemlock. ANOVA Source df ss ms F-ratio Probability Photoregime 4 4 . 08 1.02 2 . 09 0.09 8 3 Error 45 22. 00 0.49 Total 49 22. 08 183 6. ANOVA FOR TABLE 3.15: Western Hemlock Survival. ANOVA Source df ss ms F-ratio Probabi1ity Plot 4 1.53 0.38 3.14 0. 0143 Photoregime 4 1.28 0.32 2.63 0. 03 3 7 Conditioni ng 1 0.038 0. 038 0.31 0.5786 Photoregime *Conditioning 4 0.43 0.116 0.89 0.4721 Error 486 59.07 0.12 Total 499 62.34 7. ANOVA FOR TABLE 3.16: Douglas - f i r Survival. ANOVA Source df ss ms F-ratio Probability Plot 8 0.97 0.12 1.99 0. 0449 Photoregime 4 0.88 0.22 3.60 0. 0064 Conditioning 1 0.19 0.19 3.07 0.08 04 Photoregime *Conditioning 4 0.76 0.19 3.11 0. 0148 Error 882 54.06 0.061 Total 899 56.87 184 8. ANOVA FOR TABLE 3.17: Morphology of Outplanted Hemlock, i Height. ANOVA Source df ss ms F-ratio Probability Plot 4 569.80 142.45 4.15 0.0026 Photoregime 4 813.53 203.38 5.92 0. 0001 Conditioning 1 11.61 11.61 0.34 0.5613 Photoregime *Conditioning 4 229.39 57.35 1.67 0.156 0 Error 408 14010. 34.34 Total 421 15603. i i . Height Increment. ANOVA Source df ss ms F-ratio Probability Plot 4 391.28 97.82 5.65 0.0002 Photoregime 4 1215.3 3 03 . 83 17.56 0. 0000 Conditioni ng 1 27.58 27.58 1.59 0. 0275 Photoregime *Conditioning 4 54.08 13.52 0.78 0.5377 Error 410 7 09 4 .9 17.31 Total 423 8773.0 185 i i i . Caliper. ANOVA Source df ss ms F-ratio Probabili Plot 4 16.54 4.13 7.00 0. 0000 Photoregime 4 12.43 3.11 5.26 0. 0004 Conditioning 1 0.021 0.021 0. 035 0.8507 Photoregime *Conditioning 4 1.41 0.35 0.60 0.6664 Error 409 241.54 0.6 0 Total 422 272.64 i v . Relative Height Growth. ANOVA Source df ss ms F-ratio Probability Plot 4 0.76 0.19 3.40 0.0095 Photoregime 4 6.20 1.55 27.74 0.0000 Conditioning 1 0.15 0.15 2.71 0.1007 Photoregime *Conditioning 4 0.41 0.10 1.82 0.1244 Error 406 22.67 0.056 Total 419 3 0.13 9. ANOVA FOR TABLE 3.18: Morphology of Outplanted Douglas-fir. i . Height. ANOVA Source df ss ms F-ratio Probability Plot Photoregime Conditioning Photoregime *Conditioning Error Total 8 453.88 56.73 2.64 0. 0074 4 6625.3 1256.3 76.95 0.0000 1 6.92 6.92 0.32 0.57 08 4 6 03 . 70 1 50.9 3 7 . 01 0. 0 0 0 0 815 17542. 21.52 832 25243. i i . Height Increment. ANOVA Source df ss ms F-ratio Probability Plot 8 201.15 25.14 1.64 0.1104 Photoregime 4 416.06 104.02 6.77 0. 0000 Conditioning 1 37.57 37.57 2.45 0.1182 Photoregime *Conditioning 4 64.43 16.11 1.05 0.3810 Error 816 12532. 15.36 Total 833 13243. 187 i i i . Caliper. ANOVA Source df ss ms F-ratio Probabi!ity Plot 8 104. 30 13.04 4.23 0. 0000 Photoregime 4 31. 19 7.80 2.53 0. 03 9 2 Conditioning 1 0. 24 0.24 0.079 0.7786 Photoregime *Conditioning 4 9. 33 2.33 0.76 0.5535 Error 817 2517. 0 3.08 Total 834 2661. 0 iv . Relative Height Growth. ANOVA Source df ss ms F-ratio ProbabiV Plot 8 0.64 0.080 2.89 0. 0035 Photoregime 4 6.65 1.66 59.95 0. 0000 Conditioni ng 1 0. 014 0. 014 0.49 0.4842 Photoregime *Conditioning 4 0. 087 0.022 0.78 0.5374 Error 799 22.15 0. 028 Total 816 29.49 188 APPENDIX V B. CHAPTER FOUR ANOVA TABLES 1. ANOVA FOR TABLE 4.2: Terminal Bud Formation After 16 days. ANOVA Source df ss ms F-ratio Probability Induction Regime 4 5.93 1.48 7.34 0.00001 Error 220 44.40 0.20 Total 224 50.33 2. ANOVA FOR TABLE 4.3: Terminal Bud Formation After 4 weeks. ANOVA Source df ss ms F-ratio Probability Moisture stress 3 3.67 1.22 7.87 0. 00004 Photoperiod 1 15.21 15.21 97.95 0. 00000 Stress * Photoperiod 3 5.03 1.69 10.9 0 0. 00000 Error 352 54.67 0.16 Total 359 78.62 189 3. ANOVA FOR TABLE 4.4: Bud Formation and Flushing After Four and Six Weeks, i . For bud formation after four weeks see above ANOVA. i i . Flushing after 4 weeks. ANOVA Source df ss ms F-ratio Probability Moisture stress 3 4.68 1.55 12.03 0.0000 Photoperiod 1 7.22 7.23 55.77 0. 0000 Stress * Photoperiod 3 7.43 2.48 19.12 0. 0000 Error 352 45.60 0.13 Total 359 64.93 i i i . Bud Formation After Six Weeks. ANOVA Source df ss ms F-ratio Probabi1ity Moisture stress 3 4.68 1.55 12.03 0. 0000 Photoperiod 1 7.22 7.23 Stress * Photoperiod 3 7.43 2.48 19.12 0. 0000 Error 352 45.60 0.13 Total 359 64.93 190 iv . Flushing After Six Weeks. ANOVA Source df ss ms F-ratio Probability Moisture stress 3 1.56 0.52 11.79 0. 0000 Photoperiod 1 0.22 0.22 5.08 0.0248 Stress * Photoperiod 3 0.7 0 0.23 5.26 0.0000 Error 351 15.51 0. 044 Total 358 17.99 4. ANOVA FOR TABLE 4.5: Morphology Measurements, i . Height ANOVA Source df ss ms F-ratio Probability Moisture stress 3 1151.44 387.15 79.15 0. 0000 Photoperiod 1 186.91 186.91 38.21 0. 0000 Stress * Photoperiod 3 356.37 118.79 24.29 0. 0000 Error 352 1721.76 4.89 Total 359 3426.49 i i . Caliper ANOVA Source df ss ms F-ratio Probability Moisture stress 3 6.26 2.09 21.49 0. 0000 Photoperiod 1 0.10 0.10 1.07 0.3 027 Stress * Photoperiod 3 2.52 8.41 8.66 0.0000 Error 325 34.17 9.71 Total 359 192 i i i . Shoot Dry Weight ANOVA Source df ss ms F-ratio Probability Moisture stress 3 17.07 5.69 32.90 0. 0000 Photoperiod 1 1.54 1.54 8.93 0. 0032 Stress * Photoperiod 3 4.80 1.60 9.26 0.0000 Error 352 6 0.9 0 0.17 Total 359 84.33 IV. Root Dry Weight. ANOVA Source df ss ms F-ratio Probability Moisture stress 3 0.72 0.24 10.99 0. 0000 Photoperiod 1 0.63 0.63 29.04 0. 0000 Stress * Photoperiod 3 2.06 0.69 31.42 0.0000 Error 325 7.68 0.022 Total 325 11.09 193 V. Bud Height. ANOVA Source df ss ms F-ratio Probability Moisture stress 3 2.04 0.68 0.98 0.4045 Photoperiod 1 0.36 0.36 0.52 0.4798 Stress * Photoperiod 3 8.75 2.92 4.19 0. 0064 Error 352 244.82 0.69 Total 359 255.97 5. ANOVA FOR TABLE 4.10. RGC in January and March, i . January RGC. ANOVA Source df ss ms F-ratio Probability Moisture stress 3 35.93 11.98 10.66 0.0000 Photoperiod 1 0.0 0.0 0. 00 1. 0000 Stress * Photoperiod 3 24.25 8.08 7.19 0. 0001 Error 152 17 0.8 0 1.12 Total 159 230.97 i i . March RGC. ANOVA Source df ss ms F-ratio Probability Moisture stress 3 21.65 7.22 8.13 0. 0000 Photoperiod 1 0.90 0.90 1.01 0.3155 Stress * Photoperiod 3 34.55 11.52 12.48 0. 0000 Error 152 134.90 0.89 Total 159 192.00 195 6. ANOVA FOR TABLE 4:11: Dormancy Intensity in January and March, i . January Dormancy Intensity ANOVA Source df ss ms F-ratio Probability Moisture stress 3 67.36 22.45 4.64 0. 0039 Photoperiod 1 2 05 . 42 2 05 . 42 42.48 0.0000 Stress * Photoperiod 3 142.07 47.36 9.79 0.0000 Error 150 725.28 4.84 Total 157 1144.40 i i . March Dormancy Intensity ANOVA Source df ss ms F-ratio Probabi1 • Moisture stress 3 57.83 19.28 0.78 0.4973 Photoperiod 1 187 0. 00 187 0. 0 77.33 0. 0000 Stress * Photoperiod 3 301.00 100.33 4.15 0.007 4 Error 151 3651.50 24.18 Total 158 5880.6 0 APPENDIX VI DAILY MONIOTRING MEASUREMENTS OF DOUGLAS-FIR MOISTURE STRESS TRIAL MOISTURE STRESS TREATMENT DAY SHOOT WATER POTENTIAL (bars) 313 BLOCK Average Range Weight (kg) SOIL WATER CONTENT [%) Control Light Medium Severe 1 5.0 4.5 - 6.5 6.7 66 3 4.3 3.5 - 5.0 7.8 73 5 4.7 4.5 - 5.0 6.1 64 6 3.5 3.0 - 4.5 7.8 66 8 4.1 3.5 - 5.0 5.6 62 10 3.8 3.0 - 4.5 6.8 68 12 5.7 3.5 - 10.0 5.1 58 15 4.1 3.0 - 4.5 5.9 60 16 5.0 4.5 - 5.5 4.9 57 1 5.4 4.5 6. 0 6.9 69 3 4.9 4.5 - 5.5 5.8 62 5 7.5 6.5 - 10.0 4.8 49 6 9.6 7.5 - 13.5 4.5 41 8 4.0 3.5 _ 4.5 5.9 63 10 8.5 7.0 - 12.0 4.6 41 11 9.8 8.5 - 14.0 4.5 39 12 3.0 2.5 - 4.0 7.0 71 16 9.0 7.0 - 12.0 4.5 41 1 4.7 3.5 _ 5.5 7.1 70 3 5.6 4.5 - 7.0 5.8 59 5 7.3 5.5 - 8.5 4.9 61 6 9.3 7.0 - 14.0 4.6 43 7 18.4 15.0 - 2 0.0 4.3 35 8 4.9 4.0 - 6.0 7.0 71 10 4.4 3.5 - 5.0 5.5 57 12 10.5 7.0 - 13.5 4.6 41 13 16.7 12.0 - 23.5 4.4 33 1 4.5 4.0 5.0 6.9 68 3 5.8 4.5 - 7.0 5.8 63 5 7.0 5.0 - 8.5 4.9 65 6 9.9 7.5 - 15.0 4.5 40 7 17.9 12.5 - 23.5 4.2 38 8 23.8 2 0.0 - 26.0 4.0 32 10 4.0 3.0 - 5.0 6.9 68 12 3.9 3.5 - 5.0 5.6 61 14 10.8 9.0 - 15.0 4.4 43 197 APPENDIX VII DAILY MAXIMUM AND MINIMUM TEMPERATURES IN 1983 BLACK OUT SYSTEM AND ADJACENT GREENHOUSE CONTROL BENCH BLACKOUT SYSTEM GREENHOUSE CONTROL BENCH DATE " Mi nimum Maximum Maximum (1983) Temperature ( °C ) Temperature ( °C) Temperature June 22 14 22 21 23 17 25 23 24 18.5 23.5 22.5 25 18 23 21.5 26 18 27 26 27 15.5 25 25 28 15.5 23.5 22 29 15 25 25 30 14 25 21 July 1 14 21 21.5 2 13 29 25 3 14 24 24 4 16.5 28 27.5 5 13 29 25.5 6 16.5 24 23.5 7 11 23 21.5 8 11 23 23 9 15 23 21 10 14 17 19 11 14 19 18.5 12 14.5 21 20 13 13 26 26.5 14 13 27 27.5 15 13 21 19.5 16 13 26 25.5 17 14 30 30 18 14 28 28 19 14 20 21.5 20 14 21 23 21 13 28 27 22 13 31 3 0.5 23 13.5 25.5 23.5 24 13.5 28 26 25 14 29 26 26 14 21 20 27 13.5 22 21 28 13 25 23 29 13.5 29 27 30 15 30 31 31 13 29 27.5 198 APPENDIX VII DAILY MAXIMUM AND MINIMUM TEMPERATURES IN 1983 BLACK OUT SYSTEM AND ADJACENT GREENHOUSE CONTROL BENCH BLACKOUT SYSTEM GREENHOUSE CONTROL BENCH DATE '" Minimum Maximum Maximum (1983) Temperature ( °C ) Temperature ( °C) Temperature 1 August 1 14 27 24.5 2 14 21 21 3 12 24 25 4 12.5 30 27.5 5 15 27 25 6 13.5 29 28.5 7 16 32 32.5 8 13 32 34 9 15 29 27 10 14 28 23.5 11 12 23 21.5 12 13.5 27.5 26.5 13 12 30 29.5 14 12.5 28 24.5 15 12 27 25.5 14 13 27 25.3 17 25 25 

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