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The effect of nursery cultural and handling techniques on Douglas-Fir seedling quality Turner, Jennifer R. 2001

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THE EFFECT OF NURSERY CULTURAL AND HANDLING TECHNIQUES ON DOUGLAS-FIR SEEDEING QUALITY by  JENNIFER TURNER  B.Sc. Natural Resource Conservation, The University of British Columbia, 1999  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES THE FACULTY OF FORESTRY Department of Forest Sciences We accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH C O L U M B I A 2001-08-23 © Jennifer 'Turner. 2001  In  presenting  degree at the freely  this  thesis  in  partial  fulfilment  of  the  University  of  British  Columbia,  I agree  requirements that the  available for reference arid study. I further agree that  copying  of  department  this thesis or  by  his  for or  scholarly her  purposes may  representatives.  It  be is  for  an  Library shall make it  permission for extensive  granted  by the  understood  that  publication of this thesis for financial gain shall not be allowed without permission.  Department of The University of British Columbia Vancouver, Canada  DE-6 (2/88)  advanced  head  of  my  copying  or  my written  ABSTRACT This study investigated the effects of nursery cultural regime on the quality of coastal Douglas-Fir (Pseudotsuga menziesii (Mirb.) Franco var. menziesii) container seedlings. Douglas-Fir seedlings from Oregon and Vancouver Island provenances (i.e. populations) were lifted in the 1999/2000 winter on four dates, subjected to four cold storage lengths at a temperature of -2°C or +1°C, and thawed (-2°C storage only) for one or three weeks. The three-week thawing period decreased root growth capacity and chlorophyll fluorescence measures of initial survival potential below their quality thresholds. Degree-days to terminal bud break decreased with the later lift dates, longer storage lengths, and the longer thaw duration. Field survival and growth were high for all treatments tested. Only chlorophyll fluorescence was correlated with a field performance measure (relative height increment).  Up to four blackout treatments that started on one of three dates in the summer of 2000 with 10 or 20 days between multiple blackouts were tested on Douglas-Fir seedlings from a Washington provenance. A Vancouver Island provenance, given a blackout dormancy induction regime commonly used at Pelton Reforestation, was also included for comparison. Increasing the number of blackout treatments resulted in lower caliper increment, lower days to terminal bud break in winter, and higher cold hardiness on October 12. Early blackout start dates decreased the overall height increment and root growth capacity. The Vancouver Island provenance developed cold hardiness later in the fall and lost cold hardiness sooner in early spring than did the Washington provenance.  The normal lift/cold storage regime used by the nurseries does not adversely affect seedling quality. However, an extended thawing period upon cold storage removal or extended on-site storage is detrimental to seedling quality. Although measures of initial survival potential and dormancy were correlated with each other, only a weak correlation was found between initial survival potential and field performance under the field conditions in this study. Blackout regimes commonly used by British Columbia nurseries can decrease the RGC in late fall, cause quicker dormancy release and decrease the caliper.  iii  T A B L E OF CONTENTS ABSTRACT  ii  TABLE OF CONTENTS  iv  LIST OF TABLES  vii  LIST OF FIGURES  x  LIST OF APPENDICES  xiv  LIST OF ABBREVIATIONS  xvii  ACKNOWLEDGEMENTS LO PROBLEM STATEMENT  xviii 1  1.1 Objectives  3  1.2 Approach  4  2.0 LITERATURE REVIEW 2.1 Seedling Quality  5  2.2 Measures of seedling quality  6  2.2.1 Root growth capacity (RGC)  7  2.2.2 Chlorophyll fluorescence (CF)  11  2.2.3 Root electrolyte leakage (REL)  12  2.2.4 Days to terminal bud break (DBB)  13  2.3 Seedling dormancy  15  2.3.1 Pre-rest quiescence  15  2.3.2 Rest or winter dormancy  16  2.3.3 Post-dormancy quiescence  17  2.3.4 Relationship of dormancy and cold hardiness  18  2.3.5 Effects of cold storage on dormancy  20  2.4 Common handling practices from seed sow to plant  20  2.5 Dormancy induction techniques  22  2.6 Lift date  26  2.7 Cold storage  27  2.8 Thaw duration  30  2.9 Summary  32  iv  3.0 PHASE 1 - EFFECTS OF LIFT, STORE, THAW REGIMES 3.1 Phase la - Initial survival potential and dormancy  34 34  3.1.1 Objectives and Hypotheses  34  3.1.2 Methods  35  3.1.3 Results  41  3.1.4 Discussion  55  3.2 Phase lb - Field performance  65  3.2.1 Objectives and Hypotheses  65  3.2.2 Methods  66  3.2.3 Results  71  3.2.4 Discussion  80  4.0 PHASE 2- Blackout  89  4.1 Objectives and Hypotheses  90  4.2 Methods  90  4.3 Results  98  4.4 Discussion  109  4.4.1 Height growth  109  4.4.2 Caliper  109  4.4.3 Root growth capacity (RGC)  110  4.4.4 Photosynthetic efficiency after -18°C freezing (PEF)  111  4.4.5 Days to terminal bud break (DBB)  113  4.4.6 Cold Storage  115  4.4.7 Relating study results to 1999 and 2000 field observations in Oregon  116  5.0 CONCLUSION  118  6.0 RECOMMENDATIONS  121  7.0 LITERATURE CITED  122  APPENDIX I  135  APPENDIX II  137  APPENDIX III  138  v  APPENDIX IV APPENDIX V  L I S T  O F  T A B L E S  Table  Page  1.  List of treatments tested in the research greenhouse at Pelton Reforestation Ltd. for Vancouver Island and Oregon stock stored at -2°C.  35  2.  1999 Pelton Reforestation Limited handling regime for two seedlots before lift for cold storage.  36  3  Summary of statistical tests conducted on the field performance measures for Oregon and Vancouver Island provenances to ensure all treatments were tested.  41  4.  Correlation between root growth capacity (RGC), chlorophyll fluorescence (CF), and root electrolyte leakage (REL) for measuring initial survival potential, and degree-days to terminal bud break (DDBB) for measuring dormancy status. Correlation analysis was conducted on treatment means.  55  5.  Planting dates in Malcom Knapp research forest for Phase 1 treatments.  67  6.  Summary of statistical tests conducted on the field performance measures for Oregon and Vancouver Island provenances to ensure all treatments were tested.  71  7.  Least squares mean relative height increment and initial height for Oregon and Vancouver Island provenances. For test 1, data pooled for December 24, January 14, and February 4 lift dates, one and three week thawing periods, and 10 and 16-week cold storage durations. Only -2°C cold storage temperature included. For test 5, data pooled for December 24, January 14, February 4, and February 28 lift dates and one and three week thawing periods. Only 10 weeks cold storage and -2°C storage temperature included. Different letters for each test and in each column indicate a significant difference (P<0.02).  72  vii  Table  Page  8.  Least squares mean relative height increment and relative height increment per day with one and three week thawing periods. For test 1 data was pooled for December 24, January 14, and February 4 lift dates, Oregon and Vancouver Island provenances, and 10 and 16 week cold storage durations. For test 5 data was pooled for December 24, January 14, February 4, and February 28 lift dates and the two provenances. For each test and measure of height increment, different letters indicate a significant difference (P<0.01).  75  9.  Mean initial caliper for Oregon and Vancouver Island provenances. Table includes values for all tests conducted. See Table 5 for a description of the treatments pooled in each test. For each test, different letters indicate a significant difference (P<0.02).  76  10.  Least squares mean relative caliper increment and daily relative caliper increment with 10 and 16 week cold storage durations. In test 1, data pooled for December 24, January 14, and February 4 lift dates, and one and three week thawing periods. Only -2°C cold storage temperature included. In test 4, data pooled for December 24, January 14, February 4, and February 28 lift dates. Only -2°C cold storage temperature and one week thawing period included. Different letters in one column and for each test indicate a significant difference (P<0.05).  77  11.  Correlation between root growth capacity (RGC), chlorophyll fluorescence (CF), and root electrolyte leakage (REL) for measuring initial survival potential, degree-days to terminal bud break (DDBB) for measuring dormancy status, and relative height increment (RHI), relative caliper increment (RCI), frequency of multiple leaders (FML), frequency of chlorotic seedlings (FCS), and percent survival for measuring field performance. Correlation analysis was conducted on treatment means.  79  12.  List of blackout regimes conducted on the Washington provenance and tested in the agricultural greenhouse at the University of British Columbia.  91  13.  Burdett Classification System for root growth capacity (RGC).  95  viii  Summary of statistical tests for the various blackout regimes conducted on all measurement dates from October 12 to February 9 on Washington provenance Douglas-fir. Measured response variables include root growth capacity (RGC), days to terminal bud break (DBB), photosynthetic efficiency after -18°C freezing (PEF), height, and caliper. Summary of statistical tests to analyse storage and provenance differences. Measures response variables include root growth capacity (RGC), days to terminal bud break (DBB), photosynthetic efficiency after -18°C freezing (PEF), height, caliper in Test 5, and RGC, DBB, and PEF in Test 6. Least squares mean root growth capacity (RGC) and days to terminal bud break (DBB), and mean photosynthetic efficiency after -18°C freezing (PEF). Values for after cold storage and no cold storage treatments measured on March 16/2001. Values before cold storage measured on February 9/2001. Data pooled for two blackout treatments. Grouped LSMeans or grouped means with different letters are significantly different (P<0.01).  LIST OF FIGURES Figure  Page  1.  Sequential stages that occur between sowing and planting. Seedling quality can be affected at any stage, often resulting in reduced field performance after planting [adapted from Duryea (1985)].  21  2.  Least squares mean ± SE root growth capacity (RGC) for a) Oregon and b) Vancouver Island provenances for the lift-storage treatment combinations. Three-week thawing period and +1°C storage treatments not included. The threshold of 40 indicates an adequate RGC for seedlings to be planted in the field.  43  3.  Least squares mean root growth capacity (RGC) after one and three week thawing durations. Graph includes of December 24, January 14, February 4, and February 28 and 10 and 16-week storage lengths. Data pooled for Oregon and Vancouver Island provenances. +1°C storage temperature treatment not included.  44  4.  Least squares mean root growth capacity (RGC) for Oregon and Vancouver Island provenances after one and three week thawing durations. Graph includes both 10 and 16-week storage lengths. Data pooled for lift dates of December 24, January 14, February 4, and February 28. +1°C storage temperature treatment not included.  45  5.  Least squares mean ± SE root growth capacity (RGC) for Oregon and Vancouver Island provenances with storage at -2°C and +1°C. Only data from the February 4 lift date and one-week thawing period included. The threshold of 40 indicates an adequate RGC for seedlings to be planted in the field.  46  6.  Least squares mean ± SE chlorophyll fluorescence (CF) for a) Oregon and b) Vancouver Island provenances. Three-week thawing duration and +1°C storage treatments not included.  47  7.  Least squares mean chlorophyll fluorescence (CF) for four lift dates and two cold storage lengths, and one and three week thawing periods. Data pooled for Oregon and Vancouver Island provenances. Treatments with cold storage durations of 0 and 4 weeks and a storage temperature of +1°C not included.  48  x  Figure  Page  8.  Least squares mean chlorophyll fluorescence (CF) for Oregon and Vancouver Island provenances with one and three week thawing durations. Data pooled for December 24, January 14, February 4, and February 28 lift dates. Treatments with cold storage durations of 0 and 4 weeks and a storage temperature of +1°C not included.  49  9.  Least squares mean ± SE chlorophyll fluorescence (CF) for Oregon and Vancouver Island provenances with +1°C and -2°C cold storage temperatures. Only February 4 lift date and one week thaw duration treatments included.  50  10.  Least squares mean + SE degree-days to terminal bud break (DDBB) for Oregon and Vancouver Island provenances with four cold storage lengths. Data pooled for lift dates of December 24, January 14, February 4, and February 28. Three week thaw duration and +1°C storage treatments not included.  51  11.  Least squares mean ± SE degree-days to terminal bud break (DDBB) for Oregon and Vancouver Island provenances with four lift dates. Data pooled for cold storage lengths of 0, 4, 10, and 16 weeks. Three week thaw duration and +1°C storage treatments not included.  52  12.  Least squares mean ± SE degree-days to terminal bud break (DDBB) for four lift dates. Data pooled for Oregon and Vancouver Island provenances. Three-week thawing duration and +1°C storage treatments not included.  53  13.  Least squares mean degree days to terminal bud break (DDBB) for four lift dates, 10 weeks and 16 weeks cold storage, and one and three week thawing periods. Data pooled for Oregon and Vancouver Island provenances. +1°C cold storage temperature treatment not included.  54  14.  Least squares mean ± SE relative height increment with four lift dates for Oregon and Vancouver Island provenances. Data pooled for 10 and 16 week cold storage durations. Three week thawing period, +1°C cold storage temperature, and 0 and 4 weeks cold storage duration treatments not included.  73  xi  Figure  Page  15.  Least squares mean ± SE daily relative height increment with four lift dates for Oregon and Vancouver Island provenances. Data pooled for 10 and 16 week cold storage durations. Three week thawing period, +1°C cold storage temperature, and 0 and 4 weeks cold storage duration treatments not included.  74  16.  Least squares mean + SE height growth from July 24 to October 15, 2000 with three different blackout start dates. Data pooled for 10 and 20 days between blackouts and one and two blackout treatments.  99  17.  Least squares mean ± SE caliper after one, two, or three blackout treatments with 10 days between blackout treatments. Data pooled for all morphological measurement dates and the July 12 and July 26 start dates.  100  18.  Least squares mean ± SE caliper after one or two blackout treatments for the three blackout start dates. Data pooled for all morphological measurement dates and for 10 and 20 days in between blackout treatments.  100  19.  Least squares mean + SE root growth capacity (RGC) with eight measurement dates and July 12, July 26, and August 10 blackout start dates. Data pooled for one and two blackout treatments, and 10 and 20 days between blackout treatments. The threshold line indicates an adequate RGC for seedlings to be planted in the field.  101  20.  Least squares mean ± SE root growth capacity (RGC) with three blackout start dates. Data pooled for all measurement dates, for one and two blackout treatments, and for 10 and 20 days in between blackout treatments. The threshold line indicates an adequate RGC for seedlings to be planted in the field.  102  21.  Mean photosynthetic efficiency (Fv/Fm) after -18°C freezing (PEF) on the October 17 measurement date with three blackout start dates of July 12, July 26, and August 10, and one or two blackout treatments. Data pooled for 10 and 20 days between multiple blackout treatments.  104  xii  Figure  Page  22.  Mean ± SE photosynthetic efficiency (Fv/Fm) after -18°C freezing (PEF) for Washington and Vancouver Island provenances. Provenances compared for Pelton Reforestation Limited normal blackout regime. The threshold line indicates an adequate PEF for seedlings to be put into cold storage.  105  23.  Least squares mean ± SE days to terminal bud break (DBB) on January 4, January 24, and February 9, 2001, with one, two, or three blackout treatments, and 10 days in between multiple blackouts. Data pooled for blackout start dates of July 12, July 26, and Aug 10, 2000.  107  24.  Least squares mean ± SE days to terminal bud break (DBB) on January 4, January 24, and February 9, 2001 for Washington and Vancouver Island provenances subjected to Pelton Reforestation Ltd. normal blackout regime.  108  xiii  LIST OF APPENDICES Page Appendix 1  Appendix 2  Appendix 3  Table A - l . General Linear Model and statistical values for RGC. Data includes four lift dates, two cold storage lengths, two provenances, and two thawing periods.  135  Table A-2. General Linear Model and statistical values for Caliper increment. Data includes four lift dates, two cold storage lengths, and two provenances. Error split into sampling and experimental errors because 50 seedlings from each treatment were planted in rows of five.  135  Table A-3. General Linear Model and statistical values for RGC in Phase 2 study. Data includes seven test dates, three blackout start dates, one and two blackout treatments, and 10 and 20 days between blackouts. Error split into sampling and experimental errors because 20 seedlings from each treatment were planted in rows of four in the UBC agricultural greenhouse.  136  Figure A - l . Daily minimum temperature from January 1 to Oct 31 measured at the meteorological station in Malcom Knapp Research Forest.  137  Figure A-2. Daily precipitation from January 1 to Oct 31 measured at the meteorological station in Malcom Knapp Research Forest.  137  Table A-4. Summary of Oregon provenance seedling quality reported by seedling users for Pelton Reforestation Limited stock.  138  Figure A-3. Poor root development of planted Douglas-Fir seedlings produced at Pelton Reforestation (615D stocktype). Removed from Swanson Superior Forest Products field site near Noti, Oregon.  139  Figure A-4. Poor terminal shoot development of planted Douglas-Fir seedlings produced at Pelton Reforestation (615D stocktype). Removed from Swanson Superior Forest Products field site near Noti, Oregon.  139  xiv  Page Appendix 4  Table A-5. Probability values associated with Test 1 in Phase la for root growth capacity (RGC), chlorophyll fluorescence (CF), and degree-days to terminal bud break (DDBB).  140  Table A-6. Probability values or significant interactions associated with Test 2 in Phase la for root growth capacity (RGC), chlorophyll fluorescence (CF), and degree-days to terminal bud break (DDBB).  140  Table A-7. Probability values associated with Test 3 in Phase la for root growth capacity (RGC), chlorophyll fluorescence (CF), and degree-days to terminal bud break (DDBB).  140  Table A-8. Probability values associated with Test 1 in Phase lb for relative height, relative caliper, frequency of multiple leaders (FML), and frequency of chlorotic seedlings (FCS).  141  Table A-9. Probability values associated with Test 2 in Phase lb for relative height, relative caliper, frequency of multiple leaders (FML), and frequency of chlorotic seedlings (FCS).  141  Table A-10. Probability values associated with Test 3 in Phase lb for relative height, relative caliper, frequency of multiple leaders (FML), and frequency of chlorotic seedlings (FCS).  141  Table A-11. Probability values associated with Test 4 in Phase lb for relative height, relative caliper, frequency of multiple leaders (FML), and frequency of chlorotic seedlings (FCS).  142  Table A-12. Probability values associated with Test 5 in Phase lb for relative height, relative caliper, frequency of multiple leaders (FML), and frequency of chlorotic seedlings (FCS).  142  Table A-13. Probability values associated with Test 6 in Phase lb for relative height, relative caliper, frequency of multiple leaders (FML), and frequency of chlorotic seedlings (FCS).  142  xv  Page Table A-14. Probability values associated with Test 1 in Phase 2 for root growth capacity (RGC), photosynthetic efficiency after -18°C freezing (PEF), and days to terminal bud break (DBB).  Appendix 5  143  Table A-15. Probability values associated with Test 2 in Phase 2 for root growth capacity (RGC), photosynthetic efficiency after -18°C freezing (PEF), and days to terminal bud break (DBB).  143  Table A-16. Probability values associated with Test 3 in Phase 2 for root growth capacity (RGC), photosynthetic efficiency after -18°C freezing (PEF), and days to terminal bud break (DBB).  143  Table A-17. Probability values associated with Test 4 in Phase 2 for root growth capacity (RGC), photosynthetic efficiency after -18°C freezing (PEF), and days to terminal bud break (DBB).  144  Table A-18. Probability values associated with Test 5 in Phase 2 for root growth capacity (RGC), photosynthetic efficiency after -18°C freezing (PEF), and days to terminal bud break (DBB).  144  Table A-19. Probability values associated with multiple comparisons analysis in Phase 2 (Test 6) for root growth capacity (RGC), photosynthetic efficiency after -18°C freezing (PEF), and days to terminal bud break (DBB).  144  Table A-20. Least squares mean (±SE) values (relative height increment and relative caliper increment) and mean values (frequency of multiple leaders and frequency of chlorotic seedlings) for similar results with the provenance*temperature interaction in Phase lb (Test 6). Data pooled for 4 and 10 weeks cold storage.  145  Table A-21. Least squares mean (±SE) RGC values with a varying number of blackout treatments (Test 1).  145  Table A-22. Least squares mean (±SE) RGC values with a varying number of blackout treatments (Test 1).  145  xvi  LIST OF ABBREVIATIONS CF  Chlorophyll fluorescence  CH  Cold hardiness  DBB  Days to terminal bud break  DDBB  Degree-days to terminal bud break  DRI  Dormancy release index  FCS  Frequency of chlorotic seedlings  FMI  Frequency of multiple leaders  Fv/Fm  Variable fluorescence/Maximum fluorescence  GLM  General linear model  LSMean  Least Squares Mean  PEF  Photosynthetic efficiency after -18°C freezing  REL  Root electrolyte leakage  RGC  Root growth capacity  xvii  AKNOWLEDGEMENTS I would like to thank the Graduate Committee members, Dr. Steve Mitchell, Dr. Robert Guy, Dr. Steve Grossnickle, and Mr. Jolyon Hodgson, for their input and advice in all aspects of my graduate work. Financial support was provided by the Science Council of British Columbia and also by the industrial collaborator of the project, Pelton Reforestation Limited. Many of the Pelton Reforestation employees also deserve thanks for their support throughout the project. I would particularly like to recognize the assistance and advice of Fernando Ray, Mike Prueter and Dave Mac Vicar. Pelton Reforestation is also gratefully recognized for supplying all seedlings, as well as other material and equipment required for the study. Statistical advice provided by Dr. Tony Kozak and Dr. Val Lemay is recognized and appreciated.  xviii  1.0 PROBLEM STATEMENT  The environmental conditions that tree seedlings are exposed to vary throughout the year and between years. In the Pacific Northwest (southern coastal British Columbia, Washington, and Oregon), seedlings growing in the wild experience substantial changes in their environment as the seasons pass. Seedlings use environmental cues such as night length, temperature, and moisture availability to trigger the morphological and physiological changes necessary for survival and growth.  Responses to environmental cues differ among species and provenances (i.e. populations). For example, Campbell and Sorenson (1973) found a strong association between latitude of provenance for Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) and cold hardiness (CH) development in the fall. Southern provenances developed CH 1  later in the year than did northern provenances, although rate of development was similar between sources. They attributed this to differences in response to photoperiod.  Many large nurseries in British Columbia grow stock for customers from a wide geographic area, with many species and provenances. To produce high quality stock, nursery handling regimes must be tailored for each species and provenance. Pelton Reforestation Limited (Maple Ridge, British Columbia) has only recently begun  1  See List of Abbreviations for description of all abbreviations.  1  producing container stock from Oregon seed sources, as forest companies in the northwest United States have traditionally received planting stock for artificial regeneration from local bare-root nurseries. Recently, early spring planting stock grown from Oregon seed sources produced by Pelton Reforestation for their American clients exhibited poor shoot and root growth in the field (See Appendix 3, Table A-4, for detailed description of problems related to Oregon seedlots). Nursery managers wanted to understand how to tailor handling practices for southern provenance Douglas-fir that may respond to environmental cues differently than do British Columbia provenances.  In bare-root nurseries, seedlings are grown in a field outdoors. While artificial irrigation and fertilisation are common, seedlings are still exposed to ambient temperature, light intensity, and photoperiod. Seedlings grown in a container culture spend a large portion of the growing cycle in a greenhouse and growers can control the growing environment. The nursery should therefore have a detailed understanding of how environmental manipulations such as blackout and cold storage will affect the phenology, morphology, and physiology of the seedlings.  There is a large body of literature concerning the effects of nursery practices such as lift date, cold storage length, and cold storage temperature on measures of seedling quality for certain species under container culture. Thawing regimes for Picea species have also been the focus of a few recent studies. However, only one study (Rose and Haase 1997) has looked at the effect of thaw duration on Douglas-fir seedlings, and that study did not look at possible interactions between lift date, cold storage length, cold storage  2  temperature, and/or thawing duration.  Blackout, a procedure using dark curtains to artificially shorten day length, is used mostly in northern latitude areas such as British Columbia and its effect on Picea species has been sufficiently researched. Very few studies have examined blackout with coastal species or species from southern latitudes, where blackout is not often used as a dormancy induction technique. Much of the literature on blackout has focused on comparing short days and ambient day length treatments. For example, only one study (Eastham 1991) looked at the effect of blackout start date on morphological measures, but these start dates were all within a two-week period.  1.1 OBJECTIVES  The study began as an investigation into the effects of nursery handling practices, from lift date to planting date, on containerised Douglas-fir seedling quality (Phase 1). However, the completion of Phase 1 revealed the need for an additional investigation into the effects of blackout on seedling quality. The specific objectives for each Phase were as follows:  3  Phase 1 •  To ascertain the effect of lift date, cold storage duration, cold storage temperature, and thawing regime on initial seedling survival potential and field performance of northern (Vancouver Island) and southern (Oregon) provenance Douglas-fir.  •  To test correlations between different measures of seedling condition at the time of removal from cold storage, and their usefulness for predicting the field performance in the first growing season after planting.  Phase 2 •  To determine the effect of blackout regime on seedling morphology, physiology, and phenology.  1.2 APPROACH  The lift dates, storage lengths, and blackout treatments used in this study expanded on the normal timing and intensity of such treatments at the nursery. Seedling quality at lift date, or removal from storage and after one field season was assessed with commonly used response variables. In Phase 1, results for the Oregon provenance were compared with those for a Vancouver Island provenance that underwent the same treatments. In Phase 2, results were compared between a southern Washington provenance and a Vancouver Island provenance, both of which underwent the normal blackout regime at Pelton Reforestation Ltd.  4  2.0 L I T E R A T U R E R E V I E W  2.1 SEEDLING QUALITY  Seedling quality has been defined as "fitness for purpose" (Lavender et al. 1980), or the ability of a seedling to survive and grow in the field (Duryea 1985; Thompson 1985). It is important for planted seedlings to be of an acceptable quality if they are expected to perform well. Under the sub-optimal moisture, nutrient, and light conditions found on many field sites, seedlings that are healthy and vigorous when planted will have better survival and growth rates during establishment. This will minimise the need for repeated plantings or brushing treatments. Good early growth will reduce the length of time required to meet free-growing obligations and green-up requirements.  Seedlings planted in late winter and early spring are exposed to very different environmental conditions than are seedlings planted at other times of the year. Early in the season, frost events and cold soils are examples of problems that may lead to planting shock and lower field performance. Trees planted in late spring may be exposed to drought stress. Common field performance measures include first season height, caliper, and root growth. Ideally, a seedling should be in a quiescent state, cold hardy (cold hardiness, by definition, is the level of a plant's resistance to damage from low temperatures (Hobbs et al. 1992)), and have a high root growth capacity (RGC) at the time of planting.  5  2.2 MEASURES OF SEEDLING QUALITY  Many nurseries in the Pacific Northwest use only morphological specifications such as height and caliper to determine seedling quality. However, there are physiological and growth based tests.  'Initial survival potential' determines whether specific physiological processes are limited due to damage (Grossnickle and Folk 1993). 'Field performance potential' is a measure of how environmental conditions in the field will affect survival, growth, and stress resistance (Grossnickle and Folk 1993). While useful information on initial survival potential can be obtained by growing seedlings under optimal conditions, field performance potential is determined by growing seedlings in stressful environments that are closer approximations of site conditions (Folk and Grossnickle 1997).  There are a variety of tests that can be used to assess the initial survival potential or field performance potential of seedlings before they are planted. These include RGC, root electrolyte leakage (REL), and chlorophyll fluorescence (CF) (Grossnickle and Folk 1993). However, different testing techniques may give conflicting results, and there is uncertainty as to which physiological or morphological tests are the most accurate measures of initial survival potential or field performance potential.  Traditionally, actual field performance has been assessed by measuring percent survival and height increment after the first growing season (McCreary and Duryea 1987;  6  O'Reilly et al. 1999; O'Reilly et al. 2000). Dunsworth (1988) used both height and caliper (i.e. root collar diameter) increment to assess field performance of Douglas-fir and western hemlock. A less common measure for determining field performance is the frequency of multiple leaders in the field. Omi et al. (1986) found that the frequency of multiple leaders after the first growing season was negatively related to first year height growth. Another measure that may be used as an indicator of field performance is the frequency of chlorotic seedlings. Mineral deficiencies, for which chlorosis is a common visual symptom, often lead to slower growth in the field (Koslowski et al. 1991).  2.2.1 Root growth capacity (RGC)  The RGC test measures the ability of the plant to grow roots in a given period of time (7 to 28 days [Landis and Skakel 1988]) under optimal conditions (Binder et al. 1988; Grossnickle 2000). It is frequently used for measurement of initial survival potential. Roots can be grown in hydroponic systems (Ritchie 1985) or in a soil medium. Generally, the number of new roots greater than 0.5 cm or 1 cm after the specified time period are counted (Ritchie 1990). However, this type of RGC testing tends to be costly due to the labour and time required (Ritchie 1985). Other methods of calculating RGC are to sum the length of new roots or to use the displacement method where a washed root system is dipped into a 4-L container of water and the change in weight of the container due to displaced water is measured (Burdett 1979).  7  Another option is to use an index classification system that assigns a number to a range of RGC values. For example, the Burdett classification system uses six classes that range from 0 (no new root growth) to 5 (more than 30 new roots > 1 cm long) (Burdett 1979). Advantages of this method are that it is easy to perform, inexpensive relative to other RGC methods, generally repeatable, and interpretable. It is also useful to use a threshold level in RGC testing, where seedlings producing new roots above this level are predicted to perform adequately in the field (Simpson et al. 1988). An acceptable threshold for high survival in the field has been 10 new roots greater than 1 cm (Simpson et al. 1988). When counting new roots greater than 0.5 cm, 40 new roots are thought to be more than adequate (Grossnickle pers. comm.).  RGC tests can correlate well with seedling outplanting performance and survival (e.g. McTague and Tinus, 1996). RGC has commonly been cited as an accurate predictor of out-planting performance. Ritchie (1985) categorised it as possibly the most reliable method of prediction. An RGC measure (percent seedlings with new roots after 30 days in greenhouse conditions just before planting) accounted for 69% of the variation in survival of outplanted ponderosa pine {Pinus ponderosa Laws.) seedlings (Omi et al. 1994). This measure was also strongly correlated with growth in the field. Interior spruce (Picea glauca (Moench) Voss) and lodgepole pine {Pinus contorta Dougl.) seedlings producing an average RGC value of greater than 10 new roots longer than 1 cm were found to perform better in the field than those producing new roots under this threshold (Simpson et al. 1988). Lodgepole pine and white spruce seedlings from a variety of provenances were lifted at two nurseries on different dates to develop variation  8  in RGC between treatments (Burdett et al. 1983). RGC was measured before outplanting for each treatment and accounted for 85% of the variation in survival and 82% of the variation in growth between treatments.  On the other hand, the usefulness of RGC as a predictor of field performance has been repeatedly questioned. It has been described as a test of the ability to grow roots that says nothing about field survival (Binder et al. 1988). Furthermore, RGC is not a precise, accurate, or repeatable measure. It often contains within-test variation (poor precision), and will vary with different test conditions and between individuals conducting the RGC test (poor repeatability) (Binder et al. 1988). Some studies have also found the measure to have poor accuracy in its ability to predict outplanting survival (Binder et al. 1988). There are a variety of problems with using RGC to predict field performance, such as variation in the measure among individual trees and determination of the appropriate conditions and duration of the RGC test (Simpson et al. 1988). The effectiveness of RGC can also be species-dependent. Simpson et al. (1988) found a positive relationship between RGC and percent survival for interior spruce and lodgepole pine, but found no useful relationship between RGC and percent survival of interior Douglas-fir.  Landis and Skakel (1988) found that RGC tests are good predictors of initial survival potential, in that they indicate whether the seedling is alive, but not field performance potential. They suggested that CH tests, such as the -18°C test used by the BC Ministry of Forests (discussed below), or the freezing induced electrolyte leakage (FIEL) test,  9  would be more useful predictors of outplanting performance because they measure the freezing stress resistance of seedlings. For the FIEL test, leakage of electrolytes through the cell membrane is measured after seedling tissue (usually foliage, stem tissue, or both) is exposed to freezing temperatures (Burr et al. 2001).  Simpson and Ritchie (1997) wrote an article in which the two authors debated whether RGC should be used to predict field performance. Simpson argued for the effectiveness of RGC for predicting out-planting performance with the following three statements, it: "1) predicts actual field performance when trees are dead...dead trees do not grow 2)... predicts field performance potential when water uptake is dependent on new root growth, and 3) is a practical tool to monitor and improve reforestation system performance", while Ritchie argued against the proposition by stating that "1) the logic which underlies the dependence of field performance on rapid root growth after planting is flawed, i.e. root growth immediately following planting rarely occurs because soils during the planting season are generally below the threshold temperature for root growth, and 2) RGP does not provide enough information about the complex interacting factors which control field performance to give reliable, consistent predictions." (Simpson and Ritchie 1997). The article concluded with both authors admitting that there is validity to arguments for and against using RGC to predict outplanting performance.  In many cases, more information than just RGC should be incorporated (i.e. site conditions, CH, seedling morphology) before predictions of actual outplanting performance are made. If site conditions are not stressful, initial survival potential will  10  have a much stronger role in determining seedling performance in the field (Landis and Skakel 1988). However, on sites with harsh conditions, field performance is determined by an interaction between initial survival potential and site conditions.  2.2.2 Chlorophyll fluorescence (CF)  The amount of light absorbed and then emitted from a seedling's photosynthetic apparatus, termed chlorophyll fluorescence, can be used to indicate the ability of the plant to photosynthesize. That is, CF emissions are related to photosynthetic efficiency and the ability to emit unused photosynthetic energy as light (Devisscher et al. 1995). Non-variable or initial fluorescence (Fo) is fluorescence emitted when the foliage is not receiving sufficient light. Maximal fluorescence (Fm) is the maximum possible CF the needle can emit when exposed to a high light source. Variable fluorescence (Fv) is calculated by subtracting Fo from Fm. The photochemical efficiency of photosystem II is directly related to Fv/Fm.  CF is a measure of the functional integrity or the initial survival potential of a seedling (Grossnickle and Folk 1993). Damage to the photosynthetic apparatus though the application of a specific stress can be detected by a decrease in CF (Fisker et al. 1995). CF is commonly used in cold hardiness (CH) assessments to detect photosynthetic damage as a result of artificial freezing. The British Columbia Ministry of Forests measures CF in storability tests to estimate CH of seedlings at -18°C, using a threshold value of 0.65 for Fv/Fm to indicate when the low temperature has unacceptably damaged  11  seedlings (Simpson pers. comm.). Devisscher et al. (1995) found that although natural decreases in photosynthetic efficiency relating to seedling phenology did not correlate well with CH of black spruce, measuring CF was still a fast and simple measure of damage to seedlings following prolonged freezing temperatures. It should be emphasised that CF is not a direct measure of CH in seedlings.  2.2.3 Root electrolyte leakage (REL)  Electrolyte leakage occurs when cell membranes are damaged. Measures of electrolyte concentrations in bathing solutions can be used to detect damage to shoot or root tissues (Burr and Tinus 1988; McKay 1992; Folk et al. 1999). Electrolyte leakage is expressed as the ratio of the conductivities of a bathing solution before and after boiling (Folk et al. 1999). REL values greater than 30% indicate significant damage prior to excision (Grossnickle et al. 2000). It can take up to two days to run the necessary tests for REL (Burr and Tinus 1988).  REL can be a good predictor of field survival (McKay and Mason 1991). McKay (1992) found that the relationship between REL and field survival of Douglas-fir, Sitka spruce (Picea sitchensis (Bong.) Carr.), and Japanese larch (Larix leptolepis (Sieb. and Zucc.) Gord.) was negative and significant. More importantly, she found REL to be a better predictor of survival than RGC, although both tests showed significant relationships. McKay also found a relationship between REL and field survival after +1°C cold storage.  12  She suggested that cold storage caused damage to fine roots, leading to decreased survival after planting. 2.2.4 D a y s t o t e r m i n a l b u d b r e a k ( D B B )  Days to terminal bud break (DBB) or Degree-days to terminal bud break (DDBB) are indicators of dormancy. DBB is measured as the number of days it takes for seedlings grown in an optimum environment to break bud. The heat sum accumulation (DDBB), which includes both the daily temperature the plant is exposed to and the number of days until bud break, can also be used as an indicator of dormancy. Measuring the heat sum is advantageous when seedlings are grown under variable temperatures. DBB and DDBB are not measures of whether specific physiological processes are limited due to damage, and therefore do not fit into the definition of initial survival potential measures. It is, however, a measure of phenological state, and can still be a useful measure for predicting field performance potential. For example, seedlings with low dormancy (i.e. quiescent seedlings that have met a portion of their heat sum) would likely have a low field performance potential. Early dormancy release would cause the resumption of growth and a subsequent loss of water through transpiration before the formation of a sufficient root system for soil water uptake (Dunsworth 1988).  Other measures of dormancy include the Dormancy Release Index (DRI), the Oregon State University stress test, and mitotic activity in buds (Lavender 1985). DRI is the number of days to bud break for seedlings that have fully met their chilling requirement (10 typically, but it depends on seed source) divided by the average value of DBB for a  13  sample of seedlings. The Oregon State University stress test compares mortality and bud-break speed for seedlings in a controlled environment. Half of the seedlings to be tested are exposed to a temperature or moisture stress and the other half left as a control. Differences between mortality and bud break speed between the stressed and unstressed seedlings are then used to compare provenances, species, or treatments. An assumption of this test is that a lower difference in bud break between the stressed and unstressed seedlings indicates a provenance with higher vigour. Seedling vigour can be compared between provenances by determining which provenance shows the highest survival and the fastest bud break speed.  Dormant seedlings have a relatively high resistance to frost or mechanical stresses caused by harvesting seedlings at the nursery and planting them in the field (Lavender 1982). Measuring DBB is a very easy and inexpensive procedure, and has been found to correlate well with both the RGC and CH tests (e.g. Burr et al. 1989). However, unlike CH and RGC tests, DBB for a sample of seedlings must be measured over several days or weeks until all seedlings in the sample exhibit terminal bud break. RGC and CH values can be measured for the entire sample in one day.  14  2.3 SEEDLING DORMANCY  2.3.1 Pre-rest quiescence  According to a standard definition, seedling dormancy occurs when the apical meristem is predisposed to elongate or grow in some other manner, but does not do so (Doorenbos 1953). An alternate definition relates dormancy in temperate perennials to the absence of mitotic activity in the apical zone (Owens and Molder 1973). For Douglas-fir growing in Oregon, the first stage of dormancy, termed pre-rest quiescence, begins in rmdsummer when a terminal bud is formed, and ends in late September when the seedlings enter rest (Lavender 1985). At this stage, dormancy is controlled by the environment, and not by internal seedling physiology (Lavender 1985).  In nature, quiescence for Douglas-fir is usually induced by a change in photoperiod, temperature, moisture or nutrients (Kramer and Koslowksi 1979). Temperate plants are sensitive to changes in the night length. As day length shortens in mid-summer, dormancy development is signalled by the phytochrome system within the plant (Kramer and Koslowksi 1979). This triggers the cessation of shoot growth and budset that occurs with the onset of dormancy (Grossnickle 2000). Growth is inhibited with a short photoperiod by substances such as abscisic acid produced in the leaves (Kramer and Koslowski 1979).  15  Pre-rest quiescence is reversible. "Lammas" growth occurs when quiescent seedlings resume growth in the late summer or early fall after they have been exposed to a period of favourable environmental conditions (Koslowski et al. 1991). While Wareing (1985) felt that photoperiod was the most important environmental cue for triggering quiescence, Lavender (1985) noted that moisture stress is probably the major factor in many areas of the western United States prone to hot and dry summers. However, many others have determined that the most successful regime for inducing and maintaining budset is a shortened photoperiod combined with low temperature, nutrient stress, or moisture stress (Lavender et al. 1968; Macey and Arnott 1986; Simpson and Macey 1991; Landis et al. 1992).  2.3.2 Rest or winter dormancy  The second stage of dormancy, termed rest, follows quiescence and cool temperatures, and is controlled by the internal physiology of the seedling (Lavender 1991). A seedling in rest will not grow even under optimal environmental conditions. Rest can be initiated artificially with the application of abscisic acid (Kramer and Koslowski 1979). In the Pacific Northwest, rest is generally induced after seedlings are subjected to short photoperiods (Lavender 1982).  For seedlings growing in temperate climates, rest is not broken until the chilling requirement has been met (Lavender 1982). For many species, this usually occurs with exposure to temperatures around 5°C (Lavender 1981; Grossnickle 2000). Campbell and  16  Sugano (1975) confirmed this with their finding that chilling of Douglas-fir seedlings was more effective at 4.4°C than at either 7.4°C or 10°C.  Dormancy, as measured by DBB, is usually deepest in October or November and weakens as the chilling requirement of the seedling is met. van den Driessche (1975) found that for Douglas-fir, the amount of chilling required to break dormancy is approximately 12 weeks, while Wommack (1964) found the chilling requirement to be met after 8-12 weeks, depending on seed origin, at temperatures between 3°C and 6°C. Long photoperiods have also been found to break rest (Nienstaedt 1966).  2.3.3 Post-dormancy quiescence  The last stage of dormancy is called post-dormancy quiescence and, like the first stage, is controlled by the environment. Once the chilling requirement of a seedling in rest has been met, it will move into this last dormancy stage. For Douglas-fir, post-dormancy quiescence begins in mid-February and ends in early April (Lavender 1981).  Release from post-dormancy quiescence is cued mainly by warm temperatures (e.g. Lavender and Hermann 1970: Campbell and Sugano 1975; Lavender 1981). Heat sum accumulation is the sum of temperatures above a certain threshold for a particular time period and is measured in degree-days. Once chilling requirements are met, heat sum accumulation begins and eventually causes the seedling buds to flush and resume growth. Heat sum requirements vary between species and between ecotypes within species. For  17  example, northern ecotypes may begin growth after a smaller accumulation of heat sum than the more southerly ecotypes (Stern and Roche 1974). The number of chilling hours received during rest will affect heat sums. As the chilling requirement is met more fully, the heat sums needed to break bud are reduced (Lavender 1985).  2.3.4 Relationship of dormancy and cold hardiness  Cold hardiness (CH) is controlled by an interaction of the plant's genotype with the environment in which it is growing (D'Aoust and Cameron 1982). Trees become cold hardy in the winter to be able to withstand cold temperatures without suffering damage due to intracellular freezing (Levitt 1972). Growth inhibitors, such as abscisic acid, have been linked to processes causing CH development in cucumber seedlings (Rikin et al. 1976). However, the exact role of growth regulators in CH development is not known (Zwiazek et al. 2001).  In many species, development of CH is initiated by a reduction in photoperiod and coincides with growth cessation (e.g. Van den Driessche 1969; Weiser 1970; Campbell and Sugano 1975; Christersson 1978; Valkonen et al. 1990). Koslowski and Pallardy (1997) also noted that development of CH in cool temperate regions begins before the occurrence of low temperatures. The onset of near freezing to freezing temperatures induces a second stage. This stage deepens CH at a faster rate than the first stage. However, it is important to note that low temperatures alone have been shown to have little effect on CH development in Scots pine (Pinus sylvestris L.) and Norway spruce  18  (Picea abies (L.) Karst.) (Christersson 1978). Long nights in conjunction with low temperatures are more effective than treatment with only long nights (Timmis and Worrall 1975; Christersson 1978; D'Aoust and Cameron 1982).  Glerum (1976) found that maximum CH for most tree species in the northern temperate zone is attained at the beginning of December, and corresponds with the time when the tree is breaking rest by fulfilling its chilling requirement and entering post-dormancy quiescence. This coincides with the commonly held notion of CH development lagging behind dormancy development. While dormancy is (by definition) confined to the meristematic tissue of a seedling, CH affects tissues throughout the entire plant (Glerum 1976).  The biochemical changes that occur within plants with the initial development of CH are described by Koslowski and Pallardy (1997). Once bud set occurs in mid-summer, plant use of photosynthates for growth and respiration decreases, causing an accumulation of sugars early in the fall. Sugar accumulation is required for frost hardiness development. The amount of membrane lipids also increases to maintain membrane stability during cold hardiness development. To decrease the chance of intracellular ice formation, there is an increase in water-binding proteins that causes a subsequent decrease in the amount of free cellular water.  As with dormancy initiation, timing of CH development will change with species. CH development will also vary for different provenances within a species. Campbell and  19  Sorensen (1973) found that the longer the period between bud set and a frost event, the lower the level of damage due to that frost event. This same study also found that southern provenances set bud later, and therefore suffered more damage due to frost, than did seedlings from northern provenances.  2.3.5 Effects of cold storage on dormancy  Cold storage temperatures over winter are usually sufficient for partially if not fully meeting chilling requirements. Hocking and Nyland (1971) stated that storage temperatures should fall in the range of 3°C to -6°C. Others have determined that freezing cold storage temperatures are below the optimum temperature range for meeting the chilling requirement of Douglas-fir seedlings (Ritchie etal. 1985; Ritchie 1987). However, this does not mean that dormancy is not released in below-freezing storage. A temperature slightly below freezing will allow seedlings to meet their chilling requirement, but at a slower rate than would be the case if temperatures were slightly above freezing or ambient (Ritchie 1984; Ritchie 1987).  2.4 COMMON HANDLING PRACTICES FROM SEED SOWING TO PLANTING  Growing seedlings in a container nursery involves intensive manipulation and monitoring of environmental conditions in the greenhouse and outdoors. Between sowing date and lift date, seedlings are subjected to many cultural treatments in order to manipulate seedling phenology and morphology, and maximise seedling quality. From lift date to  20  planting date, handling at the nursery and/or by the seedling user can compromise seedling quality (Fig. 1).  Culturing Grading to produce 4 Lifting 4 d * Storing • seedlot Packing a n  Nurs eries  Loading 011 Shipping 4 Transporting 4 Storing 4 Planting Dock  Seedlin g Users  Figure 1. Sequential stages that occur between sowing and planting. Seedling quality can be affected at any stage, often resulting in reduced field performance after planting [adapted from Duryea (1985)]. Stock for spring planting is sown in February-March in greenhouse conditions. Depending on available nursery space in the spring, seedlings are generally moved from the greenhouse and grown outdoors. Bud set is induced around the month of July by reducing photoperiod with the objective of controlling height growth and initiating CH. The stock is typically lifted for cold storage in the late fall-early winter (Ministry of Forests 1998). Douglas-fir seedlings are normally lifted for cold storage in December or January.  An advantage of cold storage of spring stock during the winter months is that it allows the nursery to lift seedling crops in late fall and not ship until the following spring, providing the nursery with greater flexibility in seedling shipment (Rose and Haase 1997). It is feasible for coastal Douglas-fir stock to be lifted in early December, when seedlings are still dormant, and cold stored until the end of April. The maximum period  21  in cold storage is approximately 20 weeks to avoid severe depletion of carbohydrate reserves. However, not all spring planting stock is cold stored. Some coastal stock is "hot-lifted" at the end of January or early February and planted immediately.  Spring planting in the coastal biogeoclimatic zones of Vancouver Island can begin as early as February 1 (Ministry of Forests 1998), and even earlier on the Oregon coast. The BC Ministry of Forests (1998) recommends that planting in coastal zones of Vancouver Island be complete by April 15 for hot-lifted stock and April 30 for coldstored stock. However, a stock handling survey conducted for Macmillan Bloedel found that planting dates after March 15 resulted in a significantly higher incidence of failed plantations due to the lower stress resistance of stock planted after this date and the higher incidence of moisture stress (Dunsworth 1987).  2.5 DORMANCY INDUCTION TECHNIQUES  Blackout  Blackout is a common dormancy induction treatment in British Columbia nurseries. In mid to late summer, seedlings are moved into greenhouses with "blackout" capabilities. Large black curtains are suspended above and along each side of the seedlings receiving blackout to shut out light, thereby lengthening the period of darkness. The extent to which blackout is successful at inducing bud set varies with species and provenance.  22  Seedlings from low elevation or low latitude provenances require a longer night length to initiate dormancy (Tinus 1981).  Blackout induces early growth cessation of determinate species such as Douglas-fir by causing the terminal bud to set, while reducing the number and size of leaves, and root growth (Arnott and Mitchell 1981). It also stimulates the seedling to develop CH and drought resistance (Grossnickle 2000). Blackout allows the nursery to lift seedlings for cold storage at an earlier date than would be the case with natural dormancy induction (Ministry of Forests 1998).  Seedlings that receive blackout treatment generally begin growth more quickly the following spring (O'Reilly and Owens 1994). Hawkins and Hooge (1988) found that a long duration (14 hour night length) blackout resulted in earlier bud break in spruce seedlings. Little else is known about how the timing of blackout will affect dormancy release in the following growing season. One study with Sitka x white spruce hybrid seedlings did not find changes to the start date, night length, and blackout duration to have an effect on subsequent days to bud break (Eastham 1991).  Eastham (1991) noted that Sitka x white spruce hybrid seedlings that received blackout exhibited lower RGC values in the following year as compared to those that received ambient night lengths. Long night treatments have also been found to decrease the shoot:root ratio in late November due to an decrease in shoot dry weight (Eastham 1991; D'Aoust and Cameron 1982). Eastham (1991) found that longer duration blackout  23  treatments decreased the shoofcroot ratio, and that the earliest treated seedlings had the least new root growth. However, a study with spruce seedlings found that blackout did not have a negative effect on root production (Hawkins and Hooge 1988).  CH of black (Picea mariana (Mill.) B.S.P.) and white spruce was found to substantially increase five weeks after seedlings were subjected to an 8-hour photoperiod even when kept at a constant temperature of 20°C (Colombo et al. 1981). McCreary et al. (1978) looked at how CH of Douglas-fir seedlings from a Washington provenance was affected by four photoperiod lengths (6hrs, 8hrs, lOhrs, and ambient photoperiod). They found that the artificially shortened photoperiods increased the level of CH in Douglas-fir seedlings as compared to the ambient photoperiod.  One of the benefits of blackout is that can be used to produce seedlings with certain morphological specifications. Height and caliper measures are often used to grade seedlings and determine whether they are acceptable for planting (Eastham 1991). In her study with Sitka x white spruce hybrids, Eastham (1991) found that long night treatments reduced seedling height relative to ambient night lengths during the first year of growth at the Red Rock Research Station in Prince George, British Columbia. Other studies have also noted a decrease in height growth following long nights under controlled conditions (e.g. Lavender et al. 1968; D'Aoust and Cameron 1982).  24  Other Dormancy Induction Techniques  Moisture stress is a major factor in quiescence induction, particularly in regions where the climate is characteristically warm and dry (Lavender 1982). In British Columbia, moisture stress is not used by itself as a dormancy induction technique. Instead, mild moisture stress is used in concert with a blackout treatment.  To induce dormancy through moisture stress, seedlings are grown under well-irrigated conditions until the end of the growing season. At this time, seedlings are exposed to successive moisture stress treatments. At container nurseries in British Columbia, this moisture stress is achieved by drying down seedlings to about 60% of the post-irrigation container weight (Grossnickle 2000). After each treatment the seedlings are irrigated to saturation. It has been found that the severity of this treatment is just enough to cause growth cessation and not damage the seedlings. The treatments are continued in the nursery until a bud has formed. Macey and Arnott (1986) found this to be a successful technique for inducing the formation of a terminal bud in white spruce seedlings. In contrast, Carlson (1978) working with Douglas-fir concluded that moisture stress is an ineffective method of inducing budset.  The concentrations of nutrients such as nitrogen, potassium, and phosphorus can be manipulated and used to induce dormancy as well. Like moisture stress, a mild nutrient stress in conjunction with blackout has been found to be a useful technique for inducing budset (Grossnickle 2000).  25  2.6 LIFT DATE  Nursery lift dates can vary from late summer (for fall planting) to late fall/mid-winter (for late winter/early spring planting), depending on species, when seedlings are to be planted, provenance, and whether they will be cold stored. Before seedlings can be lifted for storage, the nursery must ensure that they are sufficiently cold hardy to withstand the cold temperatures in over-winter storage. Summer lifts from late June to early July occur for "hot-lifted" seedlings that will not be stored, but planted immediately. Hot lifts can also occur in mid-to-late winter for seedlings from coastal or southern provenances because there is better access to coastal or southern latitude sites at this time and because coastal/southern provenance seedlings are in post-dormancy quiescence at this time. The threat of late winter/early spring frosts is reduced on coastal or southern latitude sites as well.  Using the appropriate lifting window and proper CH techniques has been shown to substantially increase field survival potential of both Douglas-fir and ponderosa pine seedlings (Lavender and Cleary 1974). Bare-root Douglas-fir seedlings from northern California exhibited high field survival when stored in late winter, within their lifting window (Jenkinson 1984). McKay and Mason (1991) found bare-root Douglas-fir survival to increase as lift date was delayed from October to late winter. This is because seedlings are most capable of handling temperature-related stresses during the shift from rest to post-dormancy quiescence (Ritchie 1986). Seedlings that are lifted too late will  26  have resumed growth and will not be able to withstand stresses related to cold storage or planting.  2.7 COLD STORAGE  Over-winter storage of seedlings is an important practice for forest companies because it allows for an extended planting window and a longer period of employing labour associated with planting (Mattsson and Troeng 1986). Cold storage of seedlings helps to maintain CH so seedlings will be more resistant to stresses associated with planting (Ritchie et al. 1985). Although Ritchie et al. (1985) found that CH was gradually lost during storage, stored seedlings were hardier at the time of planting than were seedlings kept in the nursery bed over winter. However, seedlings lifted too early for freezer storage have shown lower root development and stomatal conductance after removal from storage due to insufficient CH (Camm et al. 1994). Cold storage has been found to reduce the RGC of both Douglas-fir and Sitka spruce (McKay and Mason 1991, Burr and Tinus 1988). Seedlings that were sufficiently cold hardy before cold storage maintained RGC in storage (Burr and Tinus 1988).  The appropriate lift date and storage duration will vary between species (McKay and Mason 1991) and provenance (Jenkinson 1984). Seedlings from high elevation sources can be stored for as long as seven months without any detrimental effects (Tung et al. 1986). In contrast, the RGC and stress resistance of Douglas-fir seedlings from lower elevation sources in Washington were found to rapidly decrease after six months cold  27  storage (Ritchie 1984; Ritchie et al. 1985). A benefit of cold storage to spring planted Douglas-fir seedlings is that dormancy release does not occur as quickly as it does under ambient temperatures (Ritchie 1984). Dormant seedlings are not as susceptible as nondormant seedlings to injuries related to early spring planting. Non-stored seedlings lifted in the spring have shown much lower stress resistance, RGC, and CH than cold stored seedlings lifted in late fall or early winter (Ritchie et al. 1985).  In British Columbia, seedlings are normally cold stored at -2°C (Van Eerden and Gates 1990). Although in the past it was common for seedlings from coastal British Columbia provenances to be stored at 2°C, even coastal seedlings are now stored at freezing temperatures (Van Eerden and Gates 1990). Van Eerden and Gates (1990) found that all o  commercial conifers grown in British Columbia could be effectively frozen stored at -2°C. It is important to note that frozen storage only freezes the soil medium around the roots (Camm et al. 1994). Temperatures around -2°C are not low enough to freeze the seedling sap (Camm et al. 1994). If seedlings are lifted at the appropriate time, when CH is maximised, they should easily withstand sub-freezing temperatures.  Nurseries in the southwest United States often use cold storage at temperatures above freezing (Burr and Tinus 1988). While there is no threat of freezing damage at these temperatures, injury due to chilling stress is still possible and the risk of grey moulds and storage moulds is increased (Camm et al. 1994; Sutherland and Hunt 1990). The rate of carbohydrate depletion is also increased for seedlings stored at temperatures above freezing due to increased respiration (Ronco 1973; Ritchie 1987; Camm et al. 1994).  28  Starch depletion during long-term storage at temperatures below freezing has been noted in ponderosa pine seedlings (Omi et al. 1994). Permanent loss of starch during low temperature storage (above or below freezing) may be caused by respiration, but temporary starch depletion can also occur with the conversion of starch to sugar (Marshall et al. 1985). The impact of carbohydrate depletion during storage may not be evident immediately upon removal from storage. Since seedlings must rely on their carbohydrate reserves to resume growth in the spring (Cannell et al. 1990), subsequent field survival may be jeopardised.  A comparison of 2°C and -4°C storage temperatures by Mattsson and Troeng (1986) found that Scots pine seedlings stored at 2°C recovered their photosynthetic abilities during the first day after removal from storage, while seedlings stored at -4°C gradually regained their photosynthetic ability, but were still substantially lower after three days. They associated this difference with the better recovery of stomatal control for the 2°C stored seedlings, resulting in less water stress immediately following planting. Overall seedling dry weight development was not different between the 2°C and -4°C storage temperatures one month after planting. Outdoor-stored seedlings, on the other hand, had a lower dry weight after one month in the field, which was attributed to root damage of outdoor-stored seedlings from low temperature exposure or root desiccation.  29  2.8 THAW DURATION  Seedlings that have been frozen stored are generally thawed before they are planted. There is debate within the literature on the appropriate thawing temperature and duration. Mattsson and Troeng (1986) found that net photosynthetic capacity, measured as CO2 exchange, of seedlings frozen at -4°C and planted without thawing was substantially less one month after planting than was the photosynthetic capacity of seedlings stored at 2°C or seedlings stored at -4°C and then thawed at 20°C for five days before planting. The current recommendation of the British Columbia Ministry of Forests is to thaw frozen container stock for 5-10 days and bareroot stock for about three days (Ministry of Forests 1998), with no temperature guidelines given. Seedlings that are thawed for five to 10 days at 10°C deplete their reserves much faster and are more susceptible to storage moulds than seedlings thawed for 5-10 days at 5°C (Silim and Guy 1998). Daniels and Simpson (1990) recommended that frozen bareroot stock be thawed at around 2°C for no more than one week so that growth of storage moulds and carbohydrate depletion can be avoided. It has also been found that frozen stock thawed too slowly will lose much of the storage reserves conserved in cold storage (Chomba 1992; Silim and Guy 1998).  One approach intermediate between thawing too slowly and not thawing at all is to subject seedlings to a rapid thaw. Silim and Guy (1998) found that lodgepole pine and interior spruce seedlings subjected to a rapid thaw at 15°C for 24-48 hrs performed as well and were more cold hardy after planting than were seedlings thawed at 2°C for two weeks. Although it took the rapidly thawed seedlings a few more days to recover their  30  photosynthetic capacity, they exhibited increased survival and less damage to the terminal bud. Camm et al. (1995) also found rapid thawing at moderate temperatures is preferable to a slow thaw at lower temperatures. They concluded that overnight, on-site thawing at ambient temperatures would sufficiently thaw Engelmann spruce seedlings for prevention of desiccation problems associated with planting frozen stock and would maintain seedling CH as well. Levesque (1995) concluded that total non-structural carbohydrates (TNC) were substantially depleted in the thawing process for both lodgepole pine and white spruce seedlings, and that longer thawing periods would result in increased TNC depletion. She also found that extended thawing accelerates dehardening and growth initiation, thereby increasing seedling susceptibility to transplant shock and late spring frosts.  Other research indicates that thawing regime does not affect seedling field performance of Douglas-fir, western larch (Larix occidentalis Nutt.), or ponderosa pine (Rose and Haase 1997). Seedlings were subjected to three different thawing regimes at 7°C after freezer storage: 1) a rapid five day thaw followed by six weeks of +1°C cold storage, 2) a six week slow thaw, and 3) an additional five weeks frozen storage followed by a one week rapid thaw. No differences were found in seedling growth or survival between the three thaw regimes. Rose and Haase (1997) still recommended that seedlings be kept frozen for as long as possible to prevent growth of storage moulds.  31  2.9 SUMMARY  Seedling quality is defined as "fitness for purpose". Many aspects of the nursery regime such as lift date, cold storage length, cold storage temperature, and dormancy induction technique can affect seedling quality. Handling by the nursery after cold storage and by the seedling user after shipment can also have an impact. Seedling quality can be monitored using measures of initial survival potential such as RGC, REL, and CF. Threshold values can be associated with each measure, below which seedling quality is unacceptable. Dormancy can also be assessed with measures such as DBB and mitotic index to get an indication of phenological state and field performance potential. If nursery regimes are properly tailored for species and provenances, initial survival potential and field performance potential should be high.  Knowledge of the appropriate lifting window is important, and damage is minimised if seedlings are lifted when dormancy and CH is high. Inappropriate cold storage length can result in low RGC and severe carbohydrate depletion. However, cold storage has been found to maintain dormancy and CH over the winter for early spring planting. Cold storage temperatures around freezing minimise carbohydrate depletion and damage to the seedlings. Knowledge of the correct thawing/on-site storage regime is important for maintaining the photosynthetic ability and carbohydrate reserves of seedlings. It is not known if interactions between thawing duration and lift date, cold storage length, or cold storage temperature could affect Douglas-fir seedling quality.  32  Blackout has been found to affect both the morphology and phenology of seedlings. Much of the research on blackout has focused on differences between long nights and ambient night lengths. The effect of multiple blackout treatments or the number of days between blackout treatments has not been studied. There is also a lack of information on how blackout affects coastal and southern conifers because it is not as commonly used in these areas as it is in northern and interior British Columbia. Drought stress alone is not a common dormancy induction technique in British Columbia, but may be used in combination with blackout.  33  3.0 P H A S E  1 - E F F E C T S  O F  L I F T ,  S T O R E ,  T  H  A  W  R E G I M E S  3.1 PHASE la - INITIAL SURVIVAL POTENTIAL AND DORMANCY  3.1.1 Objectives and Hypotheses In this study, the effects of lift, storage, and thawing regimes on seedling quality were investigated. The following hypotheses were tested: 1) Initial survival potential, as measured by RGC, CF, and REL decreases below an acceptable level due to one or more of the following treatments: a) lift dates later than December 24; b) extended cold storage up to 16 weeks; c) -2°C cold storage as compared to +1°C cold storage; d) extended thawing periods. 2) Oregon and Vancouver Island provenances are affected differently by these treatments. 3) Dormancy, as measured by degree-days to terminal bud break (DDBB), is released more quickly for: a) later lift dates; b) extended cold storage; c) +1°C cold storage as compared to -2°C cold storage; d) extended thawing periods. 4) Measures of initial survival potential and dormancy are correlated with each other.  34  3.1.2 Methods  Treatments The treatment combinations included four lift dates, four cold storage durations, two cold storage temperatures, and two thawing periods at 10°C (Table 1). The three-week thawing period was intended to represent both an extended thawing period by the nursery and/or extended on-site storage. Only seedlings stored at -2°C were used to compare one and three week thawing periods.  Table 1. List of treatments tested in the research greenhouse at Pelton Reforestation Ltd. for Vancouver Island and Oregon stock stored at -2°C. Lift  Storage Durations  Thawing Period  Dec. 24 0, 4, 10, and 16 weeks 1 and 3 weeks Jan. 14 0, 4, 10, and 16 weeks 1 and 3 weeks Feb. 4 0,4\ 10 , and 16 weeks 1 and 3 weeks Feb. 28 0, 4, 10, and 16 weeks 1 and 3 weeks for seedlings from 10 and 16 week storage treatments only also stored at +1°C with a one week thawing period a  a  b  a a  a  b  Material In order to have a sample size of 20 seedlings for each treatment combination, the study used approximately 3,020 coastal Douglas-fir seedlings (615A stocktype; i.e. a plug approximately 6 cm in diameter and 15 cm long, and grown in a Styroblock® with 45 cavities). Of these, half were from a Vancouver Island provenance (seedlot 06326:  ® Registered Trade Name of Beaver Plastics Ltd., Edmonton.  35  49°15' latitude, 123°53' longitude, 612 m elevation) and the other half from an Oregon provenance (seedlot 471-2.0: 44°10' latitude, 123°55' longitude, 330m - 660m elevation). Handling regimes at the nursery between sow date and lift date differed between the two provenances (Table 2). The spring and fall holding periods in Table 2 show the dates when the seedlings were held outdoors or in a greenhouse. The Oregon seedlot received two blackouts and was grown outdoors except when in blackout.  Table 2. 1999 Pelton Reforestation Limited handling regime for two seedlots before lift for cold storage. Provenance  Sow date  Spring holding period  Vancouver Is. 06326 seedlot Oregon 471-2.0 seedlot  Mar. 2  Mar. 2 - May 27 May 28 - Aug. 4 Feb. 10- Apr. 26 Apr. 27 - July 6  a b  Feb.10  a b a  b  1) Number of blackouts given 2) Blackout period 1) 1 blackout 2) Aug. 5 - Aug. 13 1) 2 blackouts 2) July 7 - A u g . 19  Fall holding period Aug. 14 - Oct. 26 Oct. 27 - lift date Aug. 20 - lift date  b  3  6  greenhouse outdoors  Spring stock from these two provenances is normally lifted between mid-January and mid-February. It is generally stored at -2°C for approximately four weeks, thawed for one week and then shipped in refrigerated trucks to the planting destination. Thawed seedlings are generally planted quickly with minimal on-site storage.  Sample Selection  A sample of seedlings was systematically chosen on each of the four lift dates from the operationally grown Oregon and Vancouver Island seedlots at Pelton Reforestation Ltd.  36  Due to operational constraints at the nursery, seedlings could not be randomly selected. Nursery conditions are designed to maximise spatial uniformity (Hodgson pers. comm.). It was assumed that the nursery is successful at keeping climate conditions in the greenhouse and outdoors spatially uniform for each seedlot. However, Styroblock® containers found on the edge of the greenhouse that may have received different light, temperature, and moisture conditions than those in the middle of the seedlot were not selected for the study. The seedlings used in this study were subjected to operational culling by the nursery during lifting to meet morphological specifications. Oregon provenance seedlings below a height of 18 cm, above a height of 55 cm, or below a caliper of 4.0 mm were culled. For the Vancouver Island provenance, seedlings below a height of 18 cm, above a height of 40 cm, or below a caliper of 3.5 mm were culled.  On each lift date, seedlings were removed from the Stryoblock® containers and bundled into groups of five. Seedling bundles were placed in waxed cardboard boxes lined with paper bags. Due to operational constraints, those seedling boxes placed into cold storage could not be randomly distributed throughout the cold storage facility. It was assumed that cold storage conditions were spatially uniform. Boxes that were the most accessible were removed for testing after each of the four storage lengths. For all cold storage lengths, seedling boxes were thawed in the 10°C growth chamber at the University of British Columbia and were systematically removed after a one-week thawing period. Boxes with seedlings to be used to test the three-week thawing period (after 10 and 16 weeks cold storage) remained in the 10°C growth chamber for an additional two weeks.  37  On each potting/test date, eight seedling bundles were systematically chosen from the boxes and each seedling was individually potted in the research greenhouse at Pelton Reforestation. The remainder of the seedlings were left at the University of British Columbia for REL and CF testing.  Measures of Seedling Condition and Performance  At each lift date, seedling condition was assessed using RGC, DDBB, and CF tests. Seedlings were planted for RGC testing and CF was measured on the date of lift. The same tests, with the addition of REL, were conducted to assess condition following completion of thawing. CF and REL were conducted immediately after thawing.  Root Growth Capacity (RGC)  Seedlings were potted and grown for two weeks in Pelton's research greenhouse at an average temperature of 16°C, although temperatures increased between February and July. To provide a minimum photoperiod of 14 hrs in the greenhouse throughout the year, a supplemental artificial light intensity of 40 Watts/m (total energy) was used. 2  Each treatment combination was randomly distributed in the section of the greenhouse set aside for the experiment and irrigated weekly to ensure growth occurred under optimal soil water conditions. After the two-week period, seedlings were removed from the pots and washed to expose the root system. New roots greater than 0.5 cm in length were counted. A quality threshold of 40 new roots was used because seedlings producing more than this threshold were considered to have a well functioning root system (section 2.2.1 in Literature Review).  38  Degree-days to terminal bud break (DDBB)  Seedlings were potted in Pelton's research greenhouse under the same conditions as seedlings used in RGC testing. They were grown until the day of terminal bud flush. The number of days between potting and terminal bud break was then recorded. Bud break was recorded once new foliage was visibly emerging from the terminal bud. Temperature within the greenhouse was recorded on an hourly basis with a temperature monitoring system created by Argus Control Systems Ltd. DDBB was calculated as the number of days to terminal bud flush multiplied by the average daily temperature, subtracted by 5°C; the temperature threshold commonly assumed for coastal Douglas-fir and other temperate conifer heat sum accumulation (Aitken pers. comm.)  Chlorophyll fluorescence (CF)  In order to get an indication of the photosynthetic activity of the seedling, a chlorophyll fluorometer (Opti-Sciences Inc. OS-500 Modulated Fluorometer) was used to measure variable chlorophyll 'a' fluorescence (Opti-Sciences Inc. 1995). At least four needles from each seedling were removed from various regions along the stem. The needles were dark-adapted for 10 min. by placing them in plastic clips that had a retractable window. The value for initial fluorescence (Fo) was obtained and the needles were then exposed to a flash of a high-intensity white light to determine maximal fluorescence (Fm). The threshold of 0.65 (65%) used by the Ministry of Forests in storability tests (Simpson pers. comm.) was also used as a quality threshold in this study (section 2.2.2 in Literature Review).  39  Root electrolyte leakage (REL)  Methods for conducting REL tests were obtained from Folk et al. (1999). Twenty seedlings from each provenance were thoroughly washed to remove soil from the root system and then rinsed with de-ionised water. A small section of the root system (1.5 cm x 3 cm, 1 cm depth) was removed from one side of the middle third of the root system. The section removed was large enough to cut off 20 root segments approximately 1 cm in length. Twenty root segments from each seedling were randomly selected and put into a glass tube with 10 ml of de-ionised water. The 40 tubes were placed on a shaker for 24 hrs, after which the conductivity of the solution in each tube was measured (in mhos) using a conductivity meter. Tubes were then placed in a 90°C water bath for about 15 minutes and shaken again for 20-24 hrs. REL was expressed as the ratio of initial conductivity (IC) divided by conductivity after boiling (BC), multiplied by 100 (REL (%) = IC/BC x 100). An REL value of 30% was used as a threshold to indicate significant damage prior to excision (section 2.2.3 in Literature Review).  Statistical analysis  Analysis of Variance (ANOVA) was conducted using the General Linear Model (GLM) procedure within the STAT module in SAS® for Windows (Version 6.11). Appendix 1 provides an example G L M and statistical values for Phase la, Phase lb, and Phase 2. The Least Squares Mean (LSMEAN) procedure was used to calculate the averages for each treatment because of unequal observation numbers. Due to the unbalanced design of the experiment (Table 1), three statistical tests were run for each response variable  40  because treatment combinations were grouped into three tests for analysis with the G L M procedure (Table 3). Tests of normality and homogeneity of variances conducted using SAS® indicated transformation of the data was not necessary. Probability values associated with the field measures for the three tests listed in Table 3 are given in Appendix 4. The correlation procedure was used to determine correlations between each of the response variables measured. Treatment means were used for the correlation analysis. Table 3. Summary of statistical tests conducted with the General Linear Model (GLM) procedure to ensure all treatments were tested. Lift dates  Cold Storage Cold Storage Thawing period Durations Temperatures  Test 1  Dec. 24 Jan. 14 Feb. 4 Feb. 28  0 weeks 4 weeks 10 weeks 16 weeks  -2°C  1 week  Test 2  Dec. 24 Jan.14 Feb. 4 Feb. 28 Feb. 4  10 weeks 16 weeks  -2°C  1 week 3 weeks  4 weeks 10 weeks  -2°C +1°C  1 week  Test 3  3.1.3 Results  Root growth capacity (RGC) For Test 1 that included all lift dates and cold storage durations, seedlings from all treatment combinations produced new roots above the quality threshold level (Fig. 2a and  41  2b). For Oregon seedlings that had not been stored, there was a distinct peak in RGC at the second lift date of January 14 and RGC was declining even after the February 4 lift date. For the Vancouver Island provenance, the peak in RGC for non-cold stored trees occurred on the February 4 lift date (Fig. 2b). The Oregon provenance had a significantly lower RGC than the Vancouver Island provenance (P<0.05) for 7 of the 16 treatment combinations. The RGC for the Vancouver Island provenance (P<0.01) was significantly lower for only 2 of the 16 treatment combinations, the January 14 lift with no cold storage and the February 4 lift date with no cold storage. The two provenances had similar RGC values for the December 24 and February 28 lift dates with no cold storage.  42  Figure 2. Least squares mean ± SE root growth capacity (RGC) for a) Oregon and b) Vancouver Island provenances for the lift-storage treatment combinations. Three-week thawing period and +1°C storage treatments not included. The threshold of 40 indicates an adequate RGC for seedlings to be planted in the field.  43  RGC was significantly lower for the three-week thawing period than for the one-week thawing period (P<0.01) for all lift dates for 10 and 16 weeks cold storage duration when data was pooled for Oregon and Vancouver Island provenances in Test 2 (Fig. 3). However, only seedlings that went into storage for 10 weeks on the February 28 lift and for 16 weeks on the January 14 lift had RGC values that dropped below the critical level as a result of the three-week thawing period.  120 -E? 100 o IT)  6 A O  o S (U  o O CC  24-Deci4-Jan - F e b 4  2  8-Feb  24-Dec  Cold storage length (weeks) and lift date  1 4  .  J a n  4  .  F e b  2 8  _  p e b  • 1 w eek thaw • 3 w eek thaw  Figure 3. Least squares mean root growth capacity (RGC) after one and three week thawing durations. Graph includes lift dates of December 24, January 14, February 4, and February 28 and 10 and 16-week storage lengths. Data pooled for Oregon and Vancouver Island provenances. +1°C storage treatment not included.  For both provenances with 10 and 16-week cold storage durations, and the data pooled for the four lift dates in Test 2, increasing the thawing period to three weeks significantly decreased the RGC (P<0.01) (Fig. 4). The Vancouver Island provenance had a significantly higher RGC than did the Oregon provenance (P<0.01) for both 10 and 16  44  weeks cold storage with both the one and three-week thawing periods. Although the Oregon provenance produced fewer new roots than the Vancouver Island provenance, the roots of the Oregon seedlings were substantially thicker. Longer cold storage lengths did not decrease RGC for either the one or three-week thawing periods.  120  i — —  l  Oregon  b  Oregon  10 16 Vancouver Vancouver Island  bland  • 1 w eek thaw • 3 w eek thaw  Storage Length (weeks) and Provenance  Figure 4. Least squares mean root growth capacity (RGC) for Oregon and Vancouver Island provenances after one and three week thawing durations. Graph includes both 10 and 16-week storage lengths. Data pooled for lift dates of December 24, January 14, February 4, and February 28. +1°C storage temperature treatment not included. Altering storage temperatures did not have a substantial effect on RGC in Test 3 (Fig. 5). RGC values above the quality threshold were observed for above and below freezing storage temperatures. While Oregon provenance seedlings cold stored for four weeks at +1°C produced significantly fewer new roots than those stored at -2°C, there was no difference once cold storage duration increased to 10 weeks. In contrast, when the  45  Vancouver Island provenance was cold stored for 10 weeks, R G C was significantly higher with -2°C storage than with +1°C storage.  • +1°C  • -2°C  4 Oregon  10 Oregon  4 Vancouver Is.  10 Vancouver Is.  Storage length (weeks) and Provenance  Figure 5. Least squares mean ± SE root growth capacity (RGC) for Oregon and Vancouver Island provenances with storage at -2°C and +1°C. Only data from the February 4 lift date and one-week thawing period included. The threshold of 40 indicates an adequate R G C for seedlings to be planted in the field.  Chlorophyll fluorescence (CF) Fv/Fm values never fell below the 0.65 quality threshold for either the Oregon provenance (Fig. 6a) or Vancouver Island provenance (Fig. 6b) in Test 1 for any lift date or storage length treatment with a thawing period of one week. Seedlings that did not receive cold storage maintained high Fv/Fm values within a narrow range of 0.82 and 0.85. For the Oregon provenance, cold storage durations of 10 and 16 weeks resulted in significantly lower C F than did no cold storage or four weeks cold storage for three of the four lift dates. This trend was also seen for the same lift dates with the Vancouver Island  46  provenance. However, for the January 14 lift date CF was higher with 10 and 16 weeks cold storage than with 4 weeks storage for the Oregon provenance and similar between cold storage durations for the Vancouver Island provenance.  0.5 24-Dec  14-Jan  4-Feb  28-Feb  Lift Date  B)  0.9  -r  0.8 - • — no storage  ? u.  £.  - a — 4 w eeks 0  7  - ± — 1 0 weeks  u.  -A—16 weeks  o 0.6 24-Dec 0.5  14-Jan  4-Feb  28-Feb  Lift Date  Figure 6. Least squares mean ± SE chlorophyll fluorescence (CF) for a) Oregon and b) Vancouver Island provenances. Three-week thawing duration and +1°C storage treatments not included.  47  The three-week thawing period resulted in significantly lower Fv/Fm values (P<0.001) in Test 2 for all lift dates with both 10 and 16 weeks cold storage when data was pooled for Oregon and Vancouver Island provenances (Fig. 7). CF significantly decreased (P<0.02) as storage length increased from 10 to 16 weeks with the three-week thawing period for all lift dates but February 4. For the February 4 lift date, values were similar between the two cold storage lengths (P=0.19). Values fell below the quality threshold of 0.65 with a thawing period of three weeks for the February 4 lift (10 weeks cold storage), and for the January 14, February 14, and February 28 lifts with 16 weeks cold storage. No decreasing trend in CF with later lift dates was noted for either the one or three week thawing periods.  Figure 7. Least squares mean chlorophyll fluorescence (CF) for four lift dates and two cold storage lengths, and one and three week thawing periods. Data pooled for Oregon and Vancouver Island provenances. Treatments with cold storage durations of 0 and 4 weeks and a storage temperature of+1°C not included.  48  CF significantly decreased in Test 2 with the three-week thaw duration after 10 and 16 weeks cold storage for both provenances (P<0.01) when the data was pooled for all lift dates (Fig. 8). The decrease in CF with the longer thawing duration was larger with the longer storage duration. The reduction in CF with 16 weeks storage was more pronounced with the Oregon provenance. CF dropped below the quality threshold of 0.65 with the three-week thawing duration for both provenances after 16 weeks cold storage.  E LL  "5 li. O  Oregon  Storage length (weeks)  Vancouver Is. Vancouver Is. • 1 w eek thaw and Provenance • 3 w eek thaw  Figure 8. Least squares mean chlorophyll fluorescence (CF) for Oregon and Vancouver Island provenances with one and three week thawing durations. Data pooled for December 24, January 14, February 4, and February 28 lift dates. Treatments with cold storage durations of 0 and 4 weeks and a storage temperature of + 1°C not included. In Test 3, seedlings of both provenances stored at +1°C for 4 and 10 weeks had a higher CF than did seedlings stored at a temperature of -2°C for the same duration (Fig. 9). The  49  difference in CF between storage temperatures was significantly larger with the longer storage duration of 10 weeks. The Vancouver Island provenance exhibited a larger decrease in fluorescence with -2°C cold storage for 10 weeks.  0.9  T  4 Oregon  10 Oregon  4 Vancouver Is.  10 Vancouver Is.  Storage Length (weeks) and Provenance  Figure 9. Least squares mean ± SE chlorophyll fluorescence (CF) for Oregon and Vancouver Island provenances with +1°C and -2°C cold storage temperatures. Only February 4 lift date and one week thaw duration treatments included. Degree-Days to terminal bud break (DDBB) DDBB for the Oregon and Vancouver Island provenances was similar in Test 3 between +1°C and -2°C cold storage temperatures when data for 4 and 10 weeks cold storage were pooled (LSMean = 216.36 ± 3.3 (SE) and LSMean = 217.44 ± 3.1 (SE), respectively). With the data pooled for the four lift dates in Test 1, cold storage reduced the heat sum needed to break dormancy (Fig. 10). The Oregon provenance lost dormancy sooner than the Vancouver Island provenance when cold stored for 0, 4, and  50  10 weeks. The difference in DDBB between the provenances decreased with the length of time in cold storage until 16 weeks.  Figure 10. Least squares mean ± SE degree-days to terminal bud break (DDBB) for Oregon and Vancouver Island provenances with four cold storage lengths. Data pooled for lifts dates of December 24, January 14, February 4, and February 28. Three week thaw duration and +1°C storage treatments not included. When storage lengths were pooled in Test 1, DDBB declined with lift date (Fig. 11). A significant difference between provenances in dormancy status was observed for the December 24, January 14, and February 28 lift dates.  51  550  x  500  4-  450 +  in S" 400  Oregon provenance  £ 350 | Vancouver Island provenance  0)  Q 300  m  g a  250 200 -150 100  -I  1 24-Dec  1  1 14-Jan  4-Feb  28-Feb  Lift Date  Figure 11. Least squares mean ± SE degree-days to terminal bud break (DDBB) for Oregon and Vancouver Island provenances with four lift dates. Data pooled for cold storage lengths of 0, 4, 10, and 16 weeks. Three week thaw duration and +1°C storage treatments not included. The effect of cold storage on dormancy in Test 1 was greatest for the early lift dates. DDBB decreased with the later lift dates with no cold storage when the data was pooled for the two provenances (Fig. 12). Four weeks of cold storage resulted in a significantly lower DDBB value than no cold storage for the December 24, February 4, and February 28 lift dates, but resulted in a significantly higher DDBB for the January 14 lift date. Dormancy continued to be broken while seedlings were in storage for the December 24 lift. Although dormancy continued to significantly decrease in cold storage for the February 4 lift date, the difference in DDBB between cold storage lengths was much lower than for the December 24 and January 14 lifts. DDBB significantly decreased on the February 28 lift date between no cold storage and cold storage for 4,10, or 16 weeks. However, the DDBB value with 16 weeks cold storage was significantly higher than values obtained after both 4 and 10 weeks cold storage.  52  550  T  100  -I  1  1  24-Dec  14-Jan  1  4-Feb  28-Feb  Lift Date  Figure 12. Least squares mean + SE degree-days to terminal bud break (DDBB) for four lift dates. Data pooled for Oregon and Vancouver Island provenances. Three-week thawing duration and +1°C storage treatments not included. For both 10 weeks and 16 weeks cold storage in Test 2, dormancy was generally released more rapidly when the thawing period was increased from one to three weeks (P<0.005) (Fig. 13). However, DDBB values were similar between the one and three week thawing periods on the January 14 lift date with 10 weeks cold storage (P=0.59).  53  Storage length (weeks) and Lift date  • 1 w eeks • 3 w eeks  Figure 13. Least squares mean degree days to terminal bud break (DDBB) for four lift dates, 10 weeks and 16 weeks cold storage, and one and three week thawing periods. Data pooled for Oregon and Vancouver Island provenances. +1°C cold storage temperature treatment not included.  Root electrolyte leakage (REL)  No lift, cold storage, or thaw regimes used in this study resulted in REL values above the quality threshold of 30% used to indicate damage. While significant differences were found between a few individual treatment combinations, there were no clear trends with treatment levels.  Correlations between initial survival potential and dormancy measures  Of the response variables measured, significant and positive correlations existed between RGC, DDBB, and CF (Table 4). No correlations were found between REL and RGC or DDBB. However, a weak and negative correlation existed between CF and REL (Note:  54  The correlation was negative because REL values increase with root damage while CF values decrease with damage to the photosynthetic apparatus). Table 4. Correlations between root growth capacity (RGC), chlorophyll fluorescence (CF), and root electrolyte leakage (REL) for measuring initial survival potential, and degree-days to terminal bud break (DDBB) for measuring dormancy status. Correlation analysis was conducted on treatment means. RGC  DDBB CF RGC Bi **0.4854 **0.5158 DDBB **0.4854 I **0.4399 CF **0.5158 **0.4399 REL -0.0613 -0.0285 *significant correlation with P<0.05 ** significant correlation with P<0.01  REL -0.0613 -0.0285  3.1.4 Discussion  For the one-week thawing period, no combinations of lift date or cold storage length resulted in RGC, CF, or REL values below their critical thresholds. These results allow for rejection of Hypotheses la and lb; "initial survival potential...decreases below an acceptable level [see Methods section 3.1.2 for definition of thresholds] due to...lift dates later than December 24" and "initial survival potential...decreases below an acceptable level due to...extended cold storage up to 16 weeks". Accordingly, all lift dates and cold storage lengths chosen for this study could be used in nurseries for producing coastal Douglas-fir seedlings provided quick thawing and minimal on-site storage is used.  Oregon and Vancouver Island provenances can be hot-lifted on February 4 or February 28, or lifted for cold storage between December 24 and February 28 without detrimental effects to new root growth or photosynthetic ability. This is consistent with results of Jenkinson (1984), who found that the appropriate lifting window for cold stored, low  55  elevation Oregon stock was between December and March. Low to middle elevation bare-root Douglas-fir from Washington (grown in Ireland) exhibited high survival and growth potential when lifted between January 4 and February 28 and cold stored until early June (O'Reilly et al. 1999). However, seedlings lifted on November 21 and December 19 and cold stored until early June had low survival in a year with low precipitation and high temperatures. It is important to note that these seedlings were cold stored for a longer period than the longest cold storage duration (16 weeks) used in this study.  Provided a one week thaw is used, the Oregon and Vancouver Island provenances used in this study can be cold stored for as long as 16 weeks before planting without a substantial decrease in new root growth or photosynthetic ability. Even those seedlings not removed from cold storage until July 10 had RGC values above the threshold of 40 new roots. This is consistent with the results of Ritchie (1982) and Dunsworth (1988) for Douglasfir seedlings, and Mattsson and Troeng (1986) for Scots pine seedlings. In contrast, McKay and Mason (1991) and Cannell et al. (1990) found a decrease in RGC with cold storage of Douglas-fir and Sitka spruce seedlings, although in these studies cold storage began in the fall.  A peculiar result was found for the RGC (Fig. 3), CF (Fig. 6), and DDBB (Fig. 12) measures on the January 14 lift date that did not seem to follow the trends for these measures of the other lift dates. It is unlikely that these differences for the January 14 lift date were due to the treatments. Although it was assumed that nursery conditions were  56  uniform for a particular seedlot, this may not have been the case. It is possible that some sampling error was introduced into this Phase of the study due to the systematic sampling techniques used. This explanation could also apply to other anomalies in the data mentioned in the results.  Interactions between provenance and lift date, cold storage duration, cold storage temperature, or thawing period were found for RGC, CF, and DDBB measures. This indicates that the provenances were affected differently by these treatments and hypothesis 2a, 2b, 2c, and 2d; "Oregon and Vancouver Island provenances are affected differently by a) lift dates later than December 24, b) extended cold storage, c) -2°C cold storage as compared to +1°C cold storage, and d) extended thawing periods", cannot be rejected.  The difference in DDBB between no cold storage and cold storage for 10 and 16 weeks on the February 4 and February 28 lift dates was lower than the difference observed for the December 24 and January 14 lifts. This is because the chilling requirement had been met more fully by the February 4 and February 28 lift dates. The higher DDBB values observed with 4 weeks storage as compared to no cold storage on the January 14 lift date may be due to sampling error. Differences in post-cold storage handling may have caused the upward trend for DDBB (Fig. 12) from January 14 to February 28 with 16 weeks cold storage.  57  Dormancy was released more quickly for the Oregon provenance than for the Vancouver Island provenance with 0, 4, and 10 weeks cold storage when the four lift dates were pooled (Fig. 10), and with the December 24, January 14, and February 28 lift dates when the four cold storage lengths were pooled (Fig. 11). Lower latitude provenances typically have a lower chilling requirement and begin heat sum accumulation sooner than do provenances from higher latitudes (Nienstaedt 1967). If the Oregon provenance went into dormancy in the fall sooner than the Vancouver Island provenance, this would also affect the speed with which chilling requirement was met and dormancy was released. The multiple blackout treatments given to the Oregon provenance seedlings in the summer of 1999 may have caused them to go into dormancy earlier than would be the case under natural photoperiod conditions. Phase 2 of this study investigates this possible effect of blackout regime on the phenological cycle of dormancy.  RGC and CF values decreased below the quality thresholds with the three-week thawing period for several treatments. Therefore, hypotheses Id, "Initial survival potential... decreases below an acceptable level due to...extended thawing periods", cannot be rejected. Hypotheses 3d, "Dormancy...decreases with...extended thawing periods", also cannot be rejected. The longer thawing period consistently resulted in a significant decrease in RGC, CF, and DDBB. A lower temperature or shorter thaw duration may have alleviated this problem. This is consistent with the results of Silim and Guy (1998) and Camm et al. (1995) that a rapid thaw of 24-48 hrs is the best thawing duration.  58  A thawing period of three weeks is substantially longer than the 5-10 day thawing duration recommended by the BC Ministry of Forests (Ministry of Forests 1998). Thawing at higher temperatures causes an increased loss of carbohydrates due to higher respiration rates (Levesque 1995; Silim and Guy 1998). At a thawing temperature of 10°C the accumulation of heat sum and breaking of post-dormancy quiescence occurs.  The three-week thaw was intended to simulate not only an extended thawing period at the nursery upon cold storage removal, but also improper handling via extended pre-planting storage. Even with an acceptable thawing duration, poor on-site storage could decrease stock quality so that field performance is jeopardised. On-site storage by the seedling user may have contributed to the poor root growth observed for the hot-lifted Oregon stock produced at Pelton Reforestation in 1999 and 2000. The effects of a hot lifted treatment followed by a three-week storage period (similar to the thawing period for cold stored seedlings) at 10°C was not tested in this study.  The decrease in CF with the three-week thawing period and an increase in storage length from 10 to 16 weeks (Fig. 8) indicates that there is a combined and negative effect of cold storage length and thawing period on photosynthetic ability. This trend was not apparent with RGC, indicating that this measure may not be as sensitive as CF to declines in initial survival potential before planting. Although CF and RGC are both measures of initial survival potential (Grossnickle and Folk 1993), CF is a direct measure of photosynthetic efficiency (Devisscher et al. 1995). RGC may be indirectly related to photosynthetic efficiency because of its reliance on current photosynthate (Ritchie 1982;  59  van den Driessche 1987; Binder et al. 1990; van den Driessche 1991a). RGC may have been more sensitive to the combined effect of cold storage length and thawing period had the test been conducted under more stressful conditions as has been suggested by several researchers for determining field performance potential (Folk and Grossnickle 1997; Simpson and Ritchie 1997; Grossnickle 2000). The problems of low precision (high variability between trees) and low accuracy (low predictability of seedling survival) noted for the RGC test (Binder et al. 1988; Simpson et al. 1988) may also have contributed to the inability of the measure to detect the combined effect.  CF measures photosynthetic damage or the inactivation of the photosynthetic apparatus that occurs following stress (Devisscher et al. 1995). Seedlings that are cold stored should begin photosynthetic recovery within a few hours of cold storage removal (Camm et al. 1995) and completely recover photosynthetic efficiency within a few days (Fisker et al. 1995). It is also possible that seedlings thawed for three weeks and stored for the longer duration recovered their photosynthetic ability, but to do so took longer than with the shorter thawing period of one week. The net photosynthesis of white spruce was significantly lower one day after cold storage removal for seedlings stored longer than 22 weeks (Harper and Camm 1993). However, the decrease in net photosynthesis with longterm cold storage was not evident 28 days after removal from cold storage. Fisker et al. (1995) found that RGC was high when photosynthetic ability recovered after 48 hours, but low if recovery took longer than this.  60  It was not surprising that Oregon roots were sometimes fewer but larger than Vancouver Island roots. Oregon and Vancouver Island provenances are adapted to different environmental conditions. Conditions in Oregon may favour coarse extensive root systems. It is also possible that the different root systems observed for the two provenances was due to the different cultural regimes the provenances were subjected to before lift date.  A winter peak in RGC was found in both the Vancouver Island and Oregon provenances (Fig. 2). Several studies confirm the existence of a mid-to-late winter peak in RGC (Ritchie 1982; Ritchie 1985; Cannell et al. 1990; McKay and Mason 1991; O'Reilly et al. 1999; Harper and O'Reilly 2000). Ritchie (1990) proposed that this peak exists because RGC is low when seedlings are in deep dormancy or during periods of high shoot activity during the growing season. RGC peaks occur in late summer/early autumn and in late winter/early spring when dormancy is low and shoot growth is not active. The link between RGC and dormancy may explain why the peak in RGC for the Vancouver Island provenance occurred later in the winter than the peak in RGC for the Oregon provenance. A review of RGC by Ritchie and Dunlap (1980) noted that this measure peaks once the chilling requirement has been met, and can therefore be used as an indicator of when rest is broken. Higher latitude provenances generally have a higher chilling requirement than do seedlings from southern latitudes (Neinstaedt 1967). Therefore, when grown under similar conditions, dormancy release will occur at a later date.  61  RGC for cold stored seedlings was high and did not fluctuate in the manner of non-stored seedlings. Other studies have also found that RGC remains constant when cold stored for less than 6 months (Ritchie 1982; Deans et al. 1990). A positive, statistically significant correlation was found between RGC and DBB in this study. Ritchie and Dunlap (1980) noted that RGC increases and then decreases as dormancy is gradually lost in late winter. It may be that once dormancy is released, energy is allocated more to shoot growth than root growth. However, in situations where dormancy release is not yet complete and growing conditions are favourable, energy is allocated more to the growth of roots. The winter peak was not observed in stored seedlings because dormancy release was deferred. For those seedlings stored for 10 and 16 weeks, the chilling requirement had been met and dormancy release occurred rapidly once seedlings were removed from cold storage and potted in the greenhouse.  Hypotheses 3a and 3b, "Dormancy...will decrease with...later lift dates" and "Dormancy...will decrease with...extended cold storage", cannot be rejected because DDBB decreased while in cold storage, and from the first lift on December 24 to the last lift on February 28. Seedlings not lifted until the later dates would have met their chilling requirement more fully. This corresponds well with the finding by Lavender (1981) that Douglas-fir post-dormancy quiescence does not begin until the middle of February. Cold storage temperatures around freezing are within the temperature range for meeting the chilling requirement of Douglas-fir (van den Driessche 1977; Jenkinson 1984), although these temperatures are probably below optimum for dormancy release (Ritchie 1984; Ritchie et al. 1985; Ritchie 1987; Dunsworth 1988). Seedlings in cold  62  storage did not meet their chilling requirement as quickly as those not cold stored, supporting the notion that cold storage temperatures of -2°C or +1°C are sub-optimal for meeting the chilling requirement of coastal Douglas-fir (van den Driessche 1977; Jenkinson 1984).  Both Oregon and Vancouver Island provenances could be stored at +1°C or -2°C temperatures (with a one-week thaw) without negatively affecting the initial survival potential. No differences in DDBB were found between the two cold storage temperatures. Therefore, Hypothesis lc, "Initial survival potential...decreases below an acceptable level due to...-2°C cold storage as compared to +1°C cold storage", and Hypothesis 3c "Dormancy...is released more quickly by...+l°C cold storage as compared to -2°C cold storage", can be rejected. Other studies have also found that cold storage temperatures just above or just below freezing are satisfactory (Hocking and Nyland 1971; Mattsson and Troeng 1986).  No lift date, cold storage duration, or thawing regime caused damage detectable with the REL test to the root systems of the Douglas-fir seedlings. It did not make a difference if seedlings were stored above or below freezing. REL did not increase with the threeweek thawing duration and was therefore not well correlated with either RGC or DDBB. The correlation that existed between RGC, DDBB, and CF indicates that hypothesis 4, "Measures of initial survival potential and dormancy are correlated with each other", cannot be rejected. RGC for Douglas-fir is highly dependent on the production of current photosynthate (Ritchie 1982; van den Driessche 1987; Binder et al. 1990; van den  63  Driessche 1991a), and therefore depends on how well the photosynthetic apparatus is functioning. DDBB was also correlated to RGC and CF. However, the decrease in dormancy was probably due to the accumulation of heat sum in 10°C storage, while the decrease in RGC and CF was due to a decrease in photosynthetic efficiency.  The weak, negative correlation between REL and CF is interesting because both of these measures detect damage that may have occurred to the seedling in either the roots or the photosynthetic apparatus, respectively. DDBB, RGC and CF may be better detectors of a decrease in seedling quality when seedling quality is not yet below an acceptable level. With REL, it is not until roots have been seriously damaged that a problem is detected.  64  3.2 PHASE lb - FIELD PERFORMANCE  3.2.1 Objectives a n d Hypotheses  In this study, the effects of lift, cold storage, and thawing regimes on first year field performance of Oregon and Vancouver Island provenance Douglas-fir were investigated. The following hypotheses were tested:  1) Measures of field performance of Douglas-fir from Vancouver Island and Oregon provenances after the first growing season decrease (relative height increment, relative caliper increment, survival) or increase (frequency of multiple leaders, frequency of chlorotic seedlings) due to the following treatments: a) lift dates later than December 24 b) extended cold storage c) -2°C cold storage as compared to +1°C cold storage d) extended thawing periods 2) Oregon and Vancouver Island provenances are affected differently by these treatments. 3) Measures of initial survival potential and dormancy correlate well with measures of field performance and are therefore good predictors of field performance.  65  3.2.2 Methods  Treatments A subset of treatments from the initial survival potential and dormancy study were planted for the field study (Table 5). Treatments completed in January, or treatments that were not completed until late in the growing season, were not planted. Operational planting on the coast rarely occurs at these times of year due to sub-optimal field conditions.  66  Table 5. Planting dates in Malcom Knapp research forest for Phase 1 treatments. Lift Date  Storage Length (weeks)  Thawing period (weeks)  Planting Date (2000)  Feb. 04  0  1  Feb. 07  Jan. 14  4  1  Feb. 21  Feb. 28  0  1  Feb. 28  Feb. 04  4  1  Mar. 13  Dec. 24  10  1  Mar. 13  Dec. 24  10  3  Mar. 27  Feb. 28  4  1  Apr. 3  Jan. 14  10  1  Apr. 3  Jan.14  10  3  Apr. 17  Dec. 24  16  1  Apr. 24  Feb. 04  10  1  Apr. 24  Feb. 04  10  3  May 8  Dec. 24  16  3  May 8  Jan.14  16  1  May 14  Feb. 28  10  1  May 14  Jan. 14  16  3  May 28  Feb. 28  10  3  May 28  Feb. 04  16  1  Jun. 12  Feb. 04  16  3  Jun. 26  Feb. 28  16  1  Jun. 26  Dec. 24  0  1  Not Planted  Dec. 24  4  1  Not Planted  Jan. 14  0  1  Not Planted  Feb. 28  16  3  Not Planted  67  Material As with the initial survival potential and dormancy study, a Vancouver Island provenance (seedlot 06326: 49°15' latitude, 123°53' longitude, 612m elevation) and an Oregon provenance (seedlot 471-2.0: 44°10' latitude, 123°55' longitude, 330m - 660m elevation) were used (see Table 2 for pre-lift cultural regimes).  Site Conditions An outplanting trial was established at the UBC Faculty of Forestry's Malcom Knapp Research Forest in Maple Ridge, British Columbia. Fifty seedlings from each treatment were planted in a 64m X 16m area on a well-drained, gently sloped site (southwest aspect) in the CWHdm 01 biogeoclimatic zone (285 m elevation). Large slash was manually removed to keep the site as uniform as possible. The sample trees from each treatment were planted in five rows of 10 seedlings with each row location randomly assigned within the trial area. Growing season climate records were obtained from a meteorological station located at 125m elevation (UTM co-ordinates: 531058.12W, 5456941.95 N) in the research forest. See Figures A-1 and A-2 in Appendix 2 for precipitation and average temperature in the 2000 growing season.  Sample Selection A sub-sample of seedlings was systematically selected for out planting on the date when each treatment was completed (on each lift date for those seedlings not cold stored, on each date of cold storage removal, and after both one and three week thawing periods).  68  Field performance measures  Field performance was assessed by measuring height increment, caliper (i.e. root collar diameter) increment, frequency of seedlings with multiple leaders, frequency seedlings with chlorotic foliage (trees were scored either green or chlorotic), and survival at the end of the first growing season (end of October). Caliper increment was determined by measuring initial caliper on the planting date (Table 5) and final caliper on the same seedling at the end of the growing season. Height increment was measured at the end of the growing season and initial height was determined by measuring total height and subtracting the first year height increment on the same seedling. Height and caliper increment relative to initial height and caliper were analysed after data collection because of suspected differences in initial height and initial caliper. Relative height and caliper increment per day were also analysed where applicable to account for differences due to planting date.  Statistical analysis  Analysis of Variance (ANOVA) using the General Linear Models (GLM) procedure in SAS® was used for statistical analysis of the field data. For relative height increment and relative caliper increment, Least Squares Mean (LSMean) was used to calculate the averages for each treatment because of unequal observation numbers between sampling units (rows) resulting from seedling mortality. The frequency of multiple leaders and the frequency of chlorotic seedlings required a single proportion be obtained for each row and analysis conducted on the five proportions for each treatment. Therefore, means were used to calculate the averages for each treatment for these two measures because no  69  proportion values were missing. Means were also used to calculate the averages for initial caliper with each treatment because this measure was taken on the planting date.  As with Phase la, the unbalanced design at the treatment level (Table 1) required multiple statistical tests to be run. However, the statistical tests conducted in Phase lb (Table 6) were slightly different than those used in Phase la because four treatments tested in Phase la were not planted in the field (Table 5). Probability values associated with the field measures for the six tests listed in Table 6 are given in Appendix 4. The frequency of multiple leaders and the frequency of chlorotic seedlings were log transformed because they did not meet the assumption of homogenous variances before transformation. Correlations between the response variables from the initial survival potential and dormancy study, and field performance measures from the field performance study were determined. As with the analysis in Phase la, treatment means were used to determine the correlation.  70  Table 6. Summary of statistical tests conducted on the field performance measures for Oregon and Vancouver Island provenances to ensure all treatments were tested. Lift dates  Cold Storage Cold Storage Durations Temperatures  Thawing period  Test 1  Dec. 24 Jan.14 Feb. 4  10 weeks 16 weeks  -2°C  one week three weeks  Test 2  Feb. 4 Feb. 28  0 weeks 4 weeks 10 weeks 16 weeks  -2°C  one week  Test 3  Jan. 14 Feb. 4 Feb. 28  4 weeks 10 weeks 16 weeks  -2°C  one week  Test 4  Dec. 24 Jan.14 Feb. 4 Feb. 28  10 weeks 16 weeks  -2°C  one week  Test 5  Dec. 24 Jan. 14 Feb. 4 Feb. 28  10 weeks  -2°C  one week three weeks  Test 6  Feb. 4  4 weeks 10 weeks  -2°C +1°C  one week  3.2.3 R e s u l t s  Height  Relative height increment was similar between -2°C (LSMean = 0.67 ± 0.036 (SE)) and +1°C (LSMean = 0.72 ± 0.035 (SE)) cold storage temperatures in Test 6. Provenances were not affected differently for this measure by the two temperatures (Table A-19,  71  Appendix 5). Relative height increment was higher for the Oregon provenance than for the Vancouver Island provenance in Test 1 and Test 5 when the data were pooled for one and three week thawing periods (Table 7). Initial height was similar between the Oregon and Vancouver Island provenances (e.g. test 2, LSMean = 324.24 ± 3.60 (SE) and LSMean =319.28 ± 3.61 (SE), respectively).  Table 7. Least squares mean relative height increment and initial height for Oregon and Vancouver Island provenances. For Test 1, data pooled for December 24, January 14, and February 4 lift dates, one and three week thawing periods, and 10 and 16-week cold storage durations. Only -2°C cold storage temperature included. For Test 5, data pooled for December 24, January 14, February 4, and February 28 lift dates and one and three week thawing periods. Only 10 weeks cold storage and -2°C storage temperature included. Different letters for each test and in each column indicate a significant difference (P<0.02). Provenance Test 1 Test 5  Oregon Vancouver Is. Oregon Vancouver Is.  LSMean relative height increment 0.63 0.53 0.67 0.51 a  b  a  b  LSMean initial height (mm) 303.16 319.32 313.80 312.46 a  3  a  a  A significant interaction between provenance and lift date was found in relative height increment (Fig. 14) and daily relative height increment (Fig. 15) with Test 4 when data were pooled for 10 and 16 weeks cold storage. Relative height increment was significantly lower with the February 28 lift than with the December 24 lift for the Oregon provenance. For the Vancouver Island provenance, values were similar between the four lift dates. The Oregon provenance had a significantly higher relative height increment than did the Vancouver Island provenance on the December 24 and January 14  72  lift dates. Daily relative height increment increased with lift date for the Oregon and Vancouver Island provenances. The Oregon provenance had a significantly higher daily relative height increment than did the Vancouver Island provenance on the December 24 and January 14 lift dates. The lower slope for the Oregon provenance resulted in similar daily relative height increment values for the February 4 and February 28 lift dates.  0.9  i  0.8  4  0.3 -\  , 24-Dec  , 14-Jan  , 4-Feb  28-Feb  Lift Date  Figure 14. Least squares mean ± SE relative height increment with four lift dates for Oregon and Vancouver Island provenances. Data pooled for 10 and 16-week cold storage durations. Three-week thawing period, +1°C cold storage temperature, and 0 and 4 weeks cold storage duration treatments not included  73  0.005 -, 0.0045 1 c  o  0.004 ] Oregon provenance  0.0035 ]  'Si  0) > ra  Vancouver Island provenance  0.003 •]  0.0025 i  0.002 -I  , 24-Dec  , 14-Jan  , 4-Feb  28-Feb  Lift Date  Figure 15. Least squares mean ± SE daily relative height increment with four lift dates for Oregon and Vancouver Island provenances. Data pooled for 10 and 16-week cold storage durations. Three-week thawing period, +1°C cold storage temperature, and 0 and 4 weeks cold storage duration treatments not included Although opposite trends in relative height increment and daily relative height increment were observed for the Oregon provenance, no correlation was found between relative height and the number of days in the field in the first growing season when Oregon and Vancouver Island provenances were pooled (P=0.15). However, when only the Oregon provenance was analysed, a significant correlation was found between relative height increment for each treatment and the number of days in the field (P=0.02).  Relative height increment was higher with a one week thawing period than with a three week thawing period for Test 1 when the data were pooled for December 24, January 14, and February 4 lift dates, Oregon and Vancouver Island provenances, and 10 and 16 week cold storage durations (Table 8). This difference between thawing periods was also  74  found in Test 5 with 10 weeks cold storage when data were pooled for all four lift dates. Daily relative height increment was also higher for the one-week thawing period in Test 1. However, the two thawing periods had similar daily relative height increment values for Test 5.  Table 8. Least squares mean relative height increment and relative height increment per day with one and three week thawing periods. For Test 1 data was pooled for December 24, January 14, and February 4 lift dates, Oregon and Vancouver Island provenances, and 10 and 16 week cold storage durations. For Test 5 data was pooled for December 24, January 14, February 4, and February 28 lift dates and the two provenances. For each test and measure of height increment, different letters indicate a significant difference (P<0.03).  Test 1 Test 5  Thawing period (weeks) 1 3  LSMean relative height LSMean relative increment per day height increment 0.0036 0.62 0.0033 0.53  1 3  0.0033 0.0033  3  a  b  b  3  2  0.61 0.57  a  b  Caliper  Relative caliper increment was similar between cold storage temperatures of -2°C (LSMean = 0.50 ± 0.035 (SE)) and +1°C (LSMean = 0.057 ± 0.035 (SE)) in Test 6. Provenances were not affected differently for this measure by the two cold storage temperatures (Table A-19, Appendix 5). The Oregon provenance had a significantly higher initial caliper than did the Vancouver Island provenance for all tests conducted (Table 9).  75  Table 9. Mean initial caliper for Oregon and Vancouver Island provenances. Table includes values for all tests conducted. See Table 5 for a description of the treatments pooled in each test. For each test, different letters indicate a significant difference (P<0.02). Provenance Test 1 Test 2 Test 3 Test 4 Test 5 Test 6  Oregon Vancouver Island  Mean initial caliper (mm) 6.68 5.56  Oregon Vancouver Island  6.96 5.52  Oregon Vancouver Island  6.87 5.62  Oregon Vancouver Island  6.80 5.60  Oregon Vancouver Island  6.83 5.66  Oregon Vancouver Island  6.95 5.57  a  b  a b  a b  a  b  a  b  a  b  Relative caliper increment for the Vancouver Island provenance significantly decreased as cold storage length increased from 10 to 16 weeks in Test 1 (data was pooled for December 24, January 14, and February 4 lift dates, and one and three week thawing periods) and Test 4 (data was pooled for December 24, January 14, February 4 and February 28 lift dates) (Table 10). Relative caliper increments for the Oregon provenance were similar between 10 and 16-week cold storage durations. Daily relative caliper increment was not significantly different between the two cold storage durations for the Vancouver Island provenance, but significantly increased with the longer cold storage length for the Oregon provenance (Table 10). A significant positive correlation (P<0.001) was found between the number of days in the field in the first growing season  76  and relative caliper increment when all treatments were measured including Vancouver Island and Oregon provenances.  Table 10. Least squares mean relative caliper increment and daily relative caliper increment with 10 and 16 week cold storage durations. In Test 1, data pooled for December 24, January 14, and February 4 lift dates, and one and three week thawing periods. Only -2°C cold storage temperature included. In Test 4, data pooled for December 24, January 14, February 4, and February 28 lift dates. Only -2°C cold storage temperature and one week thawing period included. Different letters in one column and for each test indicate a significant difference (P<0.05). Test Provenance 1  Oregon Vancouver Is.  4  Oregon Vancouver Is.  Storage LSMean relative LSMean relative caliper increment caliper increment per day 10 0.50 0.0026 16 0.47 0.0033 , 10 16  0.50 0.42  10 16  0.49 0.46  10 16  0.52 0.40  ab  a  ab  b  a b  ab ab  a b  0.0026 0.0029  ab ab  0.0026 0.0032  3 b  0.0027 0.0028  ab  ab  Frequency of Multiple Leaders  The frequency of multiple leaders was similar between cold storage temperatures of -2°C (Mean = 0.05 ± 0.014 (SE)) and +1°C (Mean = 0.07 + 0.017(SE)) in Test 6. Provenances were not affected differently for this measure by the two cold storage temperatures (Table A-19, Appendix 5).  77  A significant interaction between provenance and thawing period (P=0.01) was found with Test 1 when the log transformed data were pooled for lift dates of December 24, January 14, and February 4, and cold storage lengths of 10 and 16 weeks. The frequency of multiple leaders was similar between provenances with the one-week thawing period (Mean = 0.09± 0.015(SE) and Mean = 0.07± 0.016(SE), respectively). However, the Oregon provenance had a significantly higher frequency (Mean = 0.11 ± 0.017 (SE)) than did the Vancouver Island provenance (Mean = 0.04 ±0.017 (SE)) when the longer thawing period of three weeks was tested.  Frequency of chlorotic seedlings The frequency of chlorotic seedlings was similar between cold storage temperatures of -2°C (Mean = 0.10 ± 0.022 (SE)) and +1°C (Mean = 0.07 ± 0.017(SE)). Provenances were not affected differently for this measure by the two cold storage temperatures (Table A-19, Appendix 5).  Significant differences in the log transformed frequency of chlorotic seedlings in Test 1 were only found between thawing period treatments of one and three weeks. A higher frequency of chlorotic seedlings was observed for the one-week thawing period as compared to the three-week thawing period when data were pooled for the first three lift dates, cold storage lengths of 10 and 16 weeks, and Oregon and Vancouver Island provenances (Mean = 0.10 + 0.011 (SE) and 0.4 ± 0.012 (SE) respectively).  78  Correlations between Phase la and Phase lb measures  Although differences between treatments in measures of initial survival potential, dormancy and field performance were found, first year survival of all treatments was similar and high at approximately 90%. No correlations were observed between the measures of initial survival potential and dormancy, and relative caliper increment, frequency of multiple leaders, frequency of chlorotic seedlings, or survival (Table 11). However a weak positive correlation between chlorophyll fluorescence and relative height increment was observed between the treatment means.  Table 11. Correlation between root growth capacity (RGC), chlorophyll fluorescence (CF), and root electrolyte leakage (REL) for measuring initial survival potential, degreedays to terminal bud break (DDBB) for measuring dormancy status, and relative height increment (RHI), relative caliper increment (RCI), frequency of multiple leaders (FML), frequency of chlorotic seedlings (FCS), and percent survival for measuring field performance. Correlation analysis was conducted on treatment means. RHI RCI FML FCS Survival RGC DBB CF REL *significant  -0.02 0.28 0.31 0.04 0.19 *0.38 -0.085 -0.22 correlation to P<0.05  -0.26 0.0040 0.026 0.18  -0.19 0.036 0.017 0.0062  0.04 0.11 -0.16 0.011  79  3.2.4 Discussion Cold storage temperature did not limit field performance, and Hypotheses lc: "Field performance of Douglas-fir from Vancouver Island and Oregon provenances after the first growing season decreases (height increment, caliper increment, survival) or increases (frequency of multiple leaders, frequency of chlorotic seedlings) due to ...c) 2°C cold storage as compared to +1°C cold storage", can be rejected. All measures of field performance gave similar values for +1°C and -2°C cold storage temperature treatments.  Hypothesis la, "field performance of Douglas-fir from Vancouver Island and Oregon provenances after the first growing season decreases (height increment, caliper increment, survival) or increases (frequency of multiple leaders, frequency of chlorotic seedlings) due to...lift dates later than December 24", cannot be rejected because relative height increment was significantly lower for the February 28 lift date than it was for the December 24 lift date for the Oregon provenance (Fig. 14).  Daily relative height increment significantly increased for the Oregon provenance between December 24 and February 28 (Fig. 15), indicating that planting date may have caused the difference in relative height increment. Seedlings that were stored for a longer period of time were not planted until a later date (Table 5) and therefore had less time to establish and develop in the first growing season, but they grew faster per day. However, daily relative height increment can only support, not confirm, that the lower relative height increment with longer cold storage durations was due to planting date.  80  This is because daily relative height increment is also affected by planting date. Seedlings planted in early February would take longer to meet the heat sum requirement for breaking post-dormancy quiescence than those planted later in the year due to cooler temperatures at that time. Planting date likely caused the increasing trend seen for Vancouver Island and Oregon provenances for daily relative height increment since those seedlings planted later in the year would experience more favourable daily climate conditions. The explanation that planting date resulted in lower relative height increments with long cold storage lengths is further supported by the correlation that existed between relative height increment with the various treatments for the Oregon provenance, and planting date. It was not surprising that no correlation was found when the Vancouver Island provenance was included, since treatment values were similar between the four lift dates for this provenance.  Hypothesis lb, "Field performance increases or decreases due to...extended cold storage", cannot be rejected because relative caliper increment was significantly lower for 16 weeks cold storage than it was for 10 weeks cold storage for the Vancouver Island provenance (Table 10). The correlation that existed between caliper increment and days on the field site indicates that planting date may have caused the difference in relative caliper increment with cold storage length. Daily relative caliper increment was similar between the two cold storage durations, also indicating that the decrease may have been due to differences in planting date. However, the similarity in daily relative caliper increment supports but does not necessarily confirm this explanation.  81  Hypothesis Id; "Field performance increases or decreases due to...extended thawing periods" cannot be rejected because the relative height increment with the one week thawing period was higher than that with the three week thawing period (Table 8). This result cannot be explained by planting date because the results were pooled for Vancouver Island and Oregon provenances, and no correlation was found in relative height increment and days in the field for the first growing season when both provenances were included. In addition, daily relative height increment and relative height increment in Test 1 significantly decreased as thawing period increased from one to three weeks. If planting date had caused the difference, the daily relative height increment should have increased. The differences in increment between the two thawing periods were small for Test 1 (15%) and Test 5 (7%) and may not be of practical significance.  Hypotheses 2c; "Oregon and Vancouver Island provenances are affected differently by ...-2°C cold storage as compared to +1°C cold storage", can be rejected because no interaction between provenance and cold storage temperature was found for any of the field performance measures. Measures of field performance were similar between above and below freezing temperatures for both provenances.  Hypotheses 2a; "Oregon and Vancouver Island provenances are affected differently by .. .lift dates later than December 24", cannot be rejected because a significant interaction was found between provenance and lift date for relative height increment (Fig. 14). The difference between the higher daily relative height increment for the February 28 lift date  82  and the lower daily increment for the December 24 lift for the Oregon provenance was not large enough to compensate for the fact that the later lift date resulted in less growing days in the first field season. As a result, relative height increment for the Oregon provenance significantly decreased between the December 24 and February 28 lift dates. In contrast, the larger difference in daily relative height increment between the December 24 and February 28 lift dates observed for the Vancouver Island provenance may have compensated for the difference in number of growing days in the first field season between the two lift dates. This could explain why relative height increment for the Vancouver Island provenance was similar between the four lift dates.  The larger relative height increment of the Oregon provenance cannot be explained by a larger initial height, since the two provenances had similar initial height values (Table 7). This may indicate that the Oregon provenance was more suited to the field conditions than was the Vancouver Island provenance. It is also possible that genetic differences between the two provenances predisposed the Oregon seedlot to begin growth in the field earlier than the Vancouver Island seedlot after planting. Since the Oregon provenance generally had a lower dormancy than the Vancouver Island provenance in Phase la (possibly due to a more intensive blackout treatment), the Oregon provenance began growth sooner when planted in the field and had a longer growing season for when height growth could occur.  Hypothesis 2b; "Oregon and Vancouver Island provenances are affected differently by .. .extended cold storage" cannot be rejected because of the interaction found between  83  provenance and cold storage duration for relative caliper increment (Table 10). Relative caliper increment for the Oregon provenance was similar between cold storage durations of 10 and 16 weeks, while for the Vancouver Island provenance this measure significantly decreased with the longer cold storage length. This could indicate that the significant increase in daily relative caliper increment with 16 weeks cold storage was enough to compensate for the later planting date for the Oregon provenance. The similar values in daily relative caliper increment for the Vancouver Island provenance did not compensate for different planting dates, and as a result the relative caliper increment significantly decreased with the longer cold storage duration of 16 weeks.  The Oregon provenance was sown about one month earlier than the Vancouver Island provenance, and therefore had more time to grow in the first year. Along with other variations in cultural regime, this may explain why the Oregon provenance had a larger initial caliper than did the Vancouver Island provenance (Table 9). Genetic differences between the two provenances may also have resulted in different morphological or physiological responses to the cultural regime.  Hypothesis 2d; "Oregon and Vancouver Island provenances are affected differently by ...extended thawing periods", cannot be rejected because an interaction was found for the frequency of multiple leaders between provenance and thawing period. The Oregon provenance had a higher frequency of multiple leaders than did the Vancouver Island provenance when the three-week thawing period was tested. A negative correlation has been found to exist between first year height growth of Douglas-fir seedlings and the  84  frequency of multiple leaders in the field (Omi et al. 1986). In this study, however, no such correlation was found.  Frost, mechanical damage such as animal browse or vegetation press, insects, pathogens, or planting seedlings that are still in rest can all cause multiple leaders to form on planted seedlings (Lavender et al. 1990). However, these possible factors can be ruled out for this study. It is unlikely that frost damage of non-dormant buds was the cause of the higher frequency of multiple leaders in the Oregon provenance. No correlation between DDBB before planting and frequency of multiple leaders at the end of the growing season was found. Furthermore, the majority of the treatments that exhibited multiple leaders were planted after the dates on which temperatures below 0°C occurred. It is also unlikely that mechanical damage on-site caused the high frequency of multiple leaders with the Oregon provenance because there was no evidence of browsing or insect/pathogen infection at the field site.  Leader damage from extended handling before planting appears to have caused the Oregon provenance to exhibit a higher frequency of multiple leaders when thawed for three weeks. New shoots formed before planting have low stress resistance and can be easily damaged (Mitchell et al. 1990). The Oregon seedlings were generally breaking bud more rapidly than the Vancouver Island provenances in the initial survival potential and dormancy study. With the three-week thawing period, heat sum would be met at a faster rate, and the probability of new shoots forming before planting would increase.  85  Another plausible and simpler explanation may be that genetic differences between the two provenances resulted in a higher frequency of multiple leaders for the Oregon stock.  Lift date, cold storage length, and cold storage temperature did not affect the frequency of chlorotic foliage. However, a higher frequency of chlorosis was observed for a thawing period of one week as compared to three weeks. Although differences in RGC and CF were also found between the two thawing durations in the study of initial survival potential and dormancy, the higher frequency of chlorotic seedlings with the one-week thawing duration is not linked to low RGC or CF values. These two measures of initial survival potential were lower with the three-week thawing period. Chlorosis is usually caused by a nitrogen deficiency, but can also result from other nutrient deficiences or from drought stress (Koslowski and Pallardy 1997). The significantly higher daily relative height increment found in Test 1 and the earlier planting date associated with the one-week thawing period as compared to the three-week thaw may have caused seedlings to use up available nutrients and/or moisture. The difference in frequency of chlorotic seedlings (6%) is probably not of practical significance.  Relative height increment and CF exhibited a positive correlation (Table 11). Accordingly, Hypothesis 3, "Measures of initial survival potential and dormancy correlate well with measures of field performance and are therefore good predictors of field performance", cannot be rejected. Of the measures of initial survival potential and dormancy, CF exhibited the lowest percent difference (17%) between one and three week thawing periods. CF values dropped below the threshold of 0.65 with the three-week  86  thawing period in at least four treatment combinations, whereas RGC dropped below the threshold of 40 new roots <0.5cm for only two treatment combinations. The correlation between relative height increment and CF may indicate a stronger relationship between field performance and photosynthetic efficiency than between field performance and root growth capacity or dormancy status. However, it may be that CF is a better predictor of field performance than is RGC, REL, or DBB only when the negative effect of an extended thawing period is being detected.  Most of the treatments resulted in RGC, CF, and REL values above the quality thresholds. Even the few treatments that had RGC or CF values below the threshold performed well in the field. A better correlation between initial survival potential measures and field survival and growth may have been found if the treatments had resulted in a greater range of values for initial survival potential measures. This greater range may have occurred had initial survival potential been measured under stressful conditions. However, Omi et al. (1986) found that morphological measures such as height, caliper, and shoot:root ratios were consistently better predictors of field performance than were physiological measures such as budset date, bud flush date, and the OSU vigour test.  Several previous studies have found a lack of correlation between RGC and field performance (Binder et al. 1988; Landis and Skakel 1988; Simpson et al. 1988). The results from my study support conclusions of Landis and Skakel (1988) and Simpson and Ritchie (1997) that the effectiveness of RGC at predicting field performance is dependent  87  on the conditions that the seedlings are subjected to in the field. Even though the seedlings were planted on a dry site, there was relatively high rainfall throughout the 2000 growing season (Fig. A-2 in Appendix 2). Because of the mild environmental conditions in the field, differences in survival for treatments with different RGC values before planting were not apparent. Furthermore, almost all treatments with a relatively low RGC before planting were still above the threshold of 40 new roots >0.5 cm. Values above this threshold should be high enough to produce an adequate root system in the field. Values below the threshold were obtained for only two treatments; those seedlings thawed for three weeks at 10°C, lifted on February 4 and February 28 and cold stored for 10 and 16 weeks, respectively. However, even these treatments were found to perform well in the field after one growing season.  88  4.0 PHASE 2- BLACKOUT  The problems observed for the Oregon provenance as compared to the Vancouver Island provenance in Phase 1 (lower RGC, faster dormancy release, and higher incidence of multiple leaders in the field) were consistent with the problems noted for the Oregon field site in 1999 and 2000. Phase 2 investigated provenance differences to determine if they were influenced by their different blackout regimes.  Phase 2 was conducted to determine how blackout affected seedling phenology and morphology. One limitation was the inability to use the same provenances for both phases. Phase 2 used Washington and Vancouver Island provenances while Phase 1 used an Oregon provenance and a different Vancouver Island provenance.  The second phase of this study compared different blackout regimes for Douglas-fir seedlings from a Washington provenance to determine whether various blackout regimes affect dormancy release differently. Blackout has been found to cause quicker dormancy release than ambient photoperiods in western hemlock and spruce seedlings (Hawkins and Hooge 1988; O'Reilly and Owens 1989). Early dormancy release may result in increased stress during planting (Mitchell et al. 1990), reduce the rate of new root formation (Ritchie 1990), and make trees vulnerable to late frosts on field sites (Hawkins and Hooge 1988).  89  4.1 OBJECTIVES AND HYPOTHESES In this study, the effects of dormancy induction regimes on RGC, photosynthetic efficiency after -18°C freezing (PEF), and DBB at the time of lift and following cold storage were investigated. The following hypotheses were tested:  1) RGC is decreased by: a) Advancing blackout start date b) Increasing the number of blackout treatments c) Increasing the number of days between multiple blackout treatments 2) PEF is affected by: a) Advancing blackout start date b) Increasing the number of blackout treatments c) Increasing the number of days between multiple blackout treatments 3) Dormancy is released more quickly by: a) Advancing blackout start date b) Increasing the number of blackout treatments c) Increasing the number of days between multiple blackout treatments  4.2 METHODS  Experimental Treatments Combinations of three start dates, up to four blackout regimes, and two different time intervals between blackout treatments were tested (Table 12).  90  Table 12. List of blackout regimes conducted on the Washington provenance and tested in the agricultural greenhouse at the University of British Columbia. Blackout start date July 12  Number of Days between blackouts blackout treatments 1 n/a 2 10 2 20 3 10 3 20 4 10 1 July 26 n/a 2 10 2 20 3 10 1 Aug. 10 n/a 2 10 2 20 Pelton Reforestation's normal blackout regime Blackout treatments cold stored from Feb 9, 2001 to Mar 9, 2001 and grown under ambient conditions for the same time period. All blackout treatments lasted for approximately 12 days. 0  ab  b  a b  c  In order to get an indication of the effect cold storage would have on seedlings that received blackout treatment(s), two blackout treatments were put into -2°C cold storage on February 9 and removed on March 9, 2001 (Table 12). Seedlings were thawed for one week at 10°C and measured on March 16. Four weeks storage was chosen because it is common for Pelton Reforestation to cold store Douglas-fir for approximately this length of time. RGC, PEF, and DBB before and after cold storage could then be compared. The same two blackout regimes were also maintained at ambient temperatures in the greenhouse from February 9 to March 16. Non-stored seedlings could then be compared with seedlings that received cold storage for one month.  91  Material In order to have a sample size of 25 seedlings for each experimental treatment and for each measurement date, the study required approximately 2375 Douglas-fir seedlings from a Washington provenance sown on March 20/2000 (seedlot R95084: 415D stocktype, 47°42' latitude, 123°30' longitude, 244 m elevation) and 175 seedlings from a Vancouver Island provenance sown on March 16/2000 (seedlot 60641: 415D stocktype, 50°27' latitude, 125°50' longitude, 82 m elevation). Once sown, both provenances were grown in the greenhouse under ambient conditions until blackout. A Washington provenance was used because Pelton Reforestation had a surplus of seedlings for this particular seedlot, and Oregon provenance seedlings were not available. A 415D stocktype was used in this phase instead of the 615A stocktype used in Phase 1 for the same reason.  A Vancouver Island provenance that underwent a common blackout regime used at Pelton Reforestation was included for comparison with the Washington provenance that underwent the same blackout regime (Table 12). The normal blackout sequence began on July 12, ran for 12 days with a 20-day period between each of three blackouts. A l l blackout regimes used in this study ran for 12 days.  Sample Selection Six Styroblock® containers of seedlings for each treatment were set aside for the study (77 cavities/container). As with Phase 1, operational constraints required the seedling containers to be systematically chosen from the larger seedlot. Height and caliper  92  measurements were taken on five seedlings/container from the middle row of cavities. Each row contained seven seedlings. The two seedlings on either end of the row were considered buffer trees and therefore were not measured. During lifting, Washington provenance seedlings below a height of 18 cm, above a height of 45 cm, or below a caliper of 3.2 mm were culled by Pelton Reforestation Ltd. For the Vancouver Island provenance, seedlings below a height of 15 cm, above a height of 35 cm, or below a caliper of 3.2 mm were culled. To avoid the possible sampling errors encountered in Phase 1, 20 seedlings were randomly chosen from the six Styroblocks® on each measurement date for the RGC and DBB tests. Seedlings for PEF testing were randomly selected and shipped to the Ministry of Forests office in Surrey one week after the RGC and DBB measurement dates.  Measures of seedling condition  Height and caliper measurements were taken on the same 30 seedlings/experimental treatment every week from July 24 to August 28, 2000, and then every two weeks from August 28 to October 15, 2000. Starting on October 12, 2000, RGC, PEF, and DBB were measured every three weeks approximately, until February 9. The two treatments placed into cold storage on February 9 and removed on March 9, and the same two treatments that were also hot-lifted on March 16, were all tested on March 16. At each measurement date, seedling condition was assessed by measuring DBB and RGC (Burdett classification system [Table 13]) on 20 seedlings/experiment treatment. Measurement dates for five seedlings/experiment treatment to be used in PEF tests occurred approximately one week after measurement dates for DBB and RGC, except for  93  seedlings removed from cold storage on March 9 and thawed for one week, for which all testing occurred on March 16.  Root growth capacity (RGC) Twenty seedlings were planted in four rows of five in an enclosed section of the UBC agricultural greenhouse for two weeks in a mineral bed fertilised with 7 g Osmocote®/kg soil. (N:P:K in the proportions of 14:11:12) Rows were randomly distributed throughout the mineral bed. Temperature was kept between 13°C and 23 °C, and averaged around 19°C from Oct 1, 2000 to Apr 30, 2001.  Seedlings were exposed to ambient photoperiod lengths and light intensities. As with Phase 1, the seedlings were irrigated often to ensure growth occurred under optimal soil water conditions. After the two-week period, seedlings were carefully removed from the bed and the number of active roots longer than 1 cm were counted. Only 1 cm new root growth was counted so that the Burdett classification system (Burdett 1979) could be used (Table 13). This is a faster and simpler method than recording the total number of roots counted and has less observer variability (Landis and Skakel 1988). The quality threshold used for Phase 2 was a Burdett value of 4, or 11-30 new roots >lcm. Seedlings were immediately replanted once counting was finished. This enabled the same seedlings to be used for DBB testing.  Registered Tradename of Scotts Ltd., Maryville, Ohio.  94  Table 13. Burdett Classification System for root growth capacity (RGC). 0 1 2 3 4 5  No new root growth Some new roots, none >lcm 1-3 new roots >lcm 4-10 new roots >lcm 11-30 new roots >lcm >30 new roots >lcm  Days to terminal bud break (DBB)  The same 20 seedlings planted in four rows of five (rows randomly distributed) used to calculate RGC were used for determining DBB. After assessment of RGC at two weeks, seedlings were carefully replanted. The number of days between planting in the mineral bed and terminal bud flush were recorded for each measurement date. Any seedlings that were still alive but had not yet flushed their terminal bud by April 17 were not included in the statistical analyses of DBB. In a second analysis, the proportions of seedlings without terminal bud flush were compared between treatments and measurement dates. Because seedlings were planted in the UBC agricultural greenhouse, in a room where temperatures are held fairly constant, it was not necessary to use degree-days to calculate DBB.  Photosynthetic Efficiency after -18°C Freezing (PEF)  The -18°C test was used to measure PEF of five seedlings from each of the treatments. This test is currently used by the British Columbia Ministry of Forests to determine the phenological state of the seedling. That is, a seedling that survives a -18°C test is at the proper stage of dormancy and stress resistance to withstand frozen storage. The test was  95  conducted by Ronny Quay at the Ministry of Forests in Surrey, British Columbia on each measurement date.  The top 10 cm of the terminal shoot of five seedlings were removed and placed in waterfilled vials. The samples were then placed in a programmable chest freezer set at a temperature around 2°C, and kept at this temperature for 0.5 hrs. The freezer temperature was decreased to 0°C, and then dropped by 6°C/hr, for three hours, until the test temperature of -18°C was reached. Samples were held at this temperature for one hour, after which the freezer temperature was increased at 6°C/hr, for three hours, up to 0°C. The temperature was then increased to 2°C and the samples were left at this temperature for 0.5 hrs. The samples were then removed and the vials placed in a 25°C, full light growth chamber for 8 hours. Ronny Quay at the Ministry of Forests used their EARS/PPM flourometer to measure photosynthetic damage from exposure to freezing temperatures. A value for Fv/Fm greater than 0.65, set by the Ministry of Forests as the quality threshold for Douglas-fir (Simpson pers. comm.), indicated damage had occurred. While measuring PEF using CF as a measure of photosynthetic damage does not directly measure the level of cold hardiness, it can be used to determine whether differences in cold hardiness between experimental treatments exist, and which experimental treatment(s) are more cold hardy at a given point in time (Grossnickle pers. comm.).  Statistical Analyses Analysis of Variance (ANOVA) was conducted using the General Linear Model (GLM) procedure in SAS®. Least Squares Mean (LSMEAN) was used to calculate RGC and  96  DBB averages for each treatment because of unequal observation numbers between sampling units (rows). Means were used to calculate PEF averages because no observations were missing. As with Phase 1, the unbalanced design at the treatment level for Phase 2 (Table 12) required multiple statistical tests to be run (Table 14). Probability values associated with the Phase 2 measures for the six tests listed in Table 14 are given in Appendix 4. Tests of normality and homogeneity of variances indicated that data transformation was not necessary.  Table 14a. Summary of statistical tests for the various blackout regimes conducted on all measurement dates from October 12 to February 9 on Washington.provenance Douglasfir. Measured response variables include root growth capacity (RGC), days to terminal bud break (DBB), photosynthetic efficiency after -18°C freezing (PEF), height, and caliper. Blackout Number of Days in between Measured start dates blackouts blackouts response variables Test 1  July 12  1 2 3 4  10  RGC DBB PEF Height Caliper  Test 2  July 12  1 2 3  10 20  RGC DBB CH Height Caliper  Test 3  July 12 July 26  1 2 3  10  RGC DBB CH Height Caliper  97  Test 4  July 12 July 26 Aug. 10  1 2  RGC DBB CH Height Caliper  10 20  Table 14b. Surnmary of statistical tests to analyse storage and provenance differences. Measured response variables include root growth capacity (RGC), days to terminal bud break (DBB), photosynthetic efficiency after -18°C freezing (PEF), height, and caliper in Test 5, and RGC, DBB, and PEF in Test 6. Provenances  Storage length  Measurement dates  Response variables measured  Test 5  a  Vancouver Is. Washington  0 weeks  All measurement dates excluding March 16  RGC DBB CH Height Caliper  Test 6  b  Washington  0 weeks 4 weeks  February 9 March 16  RGC DBB CH  a b  Only Pelton's common blackout regime tested Only two blackout regimes tested  4.3 RESULTS  Height growth Height growth from July 24 to October 15, 2000, in Test 4 varied according to the start date of the first blackout treatment when the height data was pooled for one and two blackout treatments, and 10 and 20 days between blackout treatments (Fig. 16). Those seedlings that received the earliest blackout start date (July 12) had the lowest height growth throughout the growing period. A blackout start date of July 26 produced  98  seedlings with intermediate height growth, while those seedlings that did not receive the first blackout until August 10 had the greatest height. For all blackout start dates, there was a lag time of two to three weeks before there was a decrease in the rate of height growth.  330 -,  170 150  -I  ,  ,  ,  ,  ,  ,  8-Jul  28-Jul  17-Aug  6-Sep  26-Sep  16-Oct  5-Nov  Measurement Date  Figure 16. Least squares mean ± SE height growth from July 24 to October 15, 2000 with three different blackout start dates. Data pooled for 10 and 20 days between blackouts and one and two blackout treatments.  Caliper Caliper significantly decreased in Test 3 as the number of blackout treatments increased from one to three, when the data were pooled for the early and middle start dates (Fig. 17). Caliper was also significantly lower with two blackouts compared to one blackout treatment when all three start dates were considered in Test 4 (Fig. 18).  99  Figure 17. Least squares mean ± SE caliper after one, two, or three blackout treatments with 10 days between blackout treatments. Data pooled for all morphological measurement dates and the July 12 and July 26 start dates.  • 1 Blackout • 2 Blackouts  13-Jul  26-Jul  10-Aug  Measurement Date  Figure 18. Least squares mean ± SE caliper after one or two blackout treatments for the three blackout start dates. Data pooled for all morphological measurement dates and for 10 and 20 days in between blackout treatments.  100  Root growth capacity (RGC) In all tests RGC was similar whether 10 or 20 days between blackout treatments were used (e.g. in Test 4, LSMean = 3.70 ± 0.04 (SE) and LSMean = 3.67 ± 0.04 (SE), respectively). This measure was not affected by the number of blackout treatments used (e.g. Table A-20, Appendix 5). In Test 4, RGC values differed between the measurement dates, starting out relatively high on October 12 and decreasing to a low by November 23 (Fig. 19). As with Phase 1 of this study, a winter peak in RGC was observed. The highest RGC value was obtained on the February 9 measurement date.  6i  10  -I  ,  ,  ,  1-Oct  20-Nov  9-Jan  28-Feb  Measurement Date  Figure 19. Least squares mean ± SE root growth capacity (RGC) with eight measurement dates and July 12, July 26, and August 10 blackout start dates. Data pooled for one and two blackout treatments, and 10 and 20 days in between blackout treatments. The threshold line indicates an adequate RGC for seedlings to be planted in the field. RGC fell below a Burdett value of 4 for all but the October 12 and February 9 measurement dates. Start date had a marginally significant effect (P=0.058) in Test 4 when data for each measurement date, one and two blackout treatments, and 10 or 20  101  days in between blackout treatments were pooled (Fig. 20). The early and middle start dates gave similar RGC values, and they each had significantly lower RGC values than the late start date. Figure 19 shows that RGC for the late blackout start date had a higher minimum value on the November 23 and December 14 measurement dates.  ~ E  5  T  o  12-Jul  26-Jul  10-Aug  Blackout Start Date  Figure 20. Least squares mean ± SE root growth capacity (RGC) with three blackout start dates. Data pooled for all measurement dates, for one and two blackout treatments, and for 10 and 20 days in between blackout treatments. The threshold line indicates an adequate RGC for seedlings to be planted in the field. RGC values were similar before cold storage on February 9 and after removal from cold storage for 4 weeks at -2°C on March 16 when the data was pooled for the two treatments that received cold storage (Table 15). However, RGC significantly decreased between February 9 and March 16 if seedlings were not cold stored. RGC of non-stored seedlings was also significantly lower than that of cold stored seedlings on March 16.  102  Table 15. Least squares mean root growth capacity (RGC) and days to terminal bud break (DBB), and mean photosynthetic efficiency after -18°C freezing (PEF). Values for after cold storage and no cold storage treatments measured on March 16/2001. Values before cold storage measured on February 9/2001. Data pooled for two blackout treatments. Grouped LSMeans or grouped means with different letters are significantly different (P<0.01). Experiment treatmentRGC LSMean PEF Mean  DBB LSMean  before cold storage after cold storage no cold storage  28.70 20.74 15.81  4.46 3.90 2.55  a a b  0.82 0.83 0.46  a  a b  a b  c  Photosynthetic efficiency after -18°C freezing (PEF)  In all tests PEF was not affected by whether 10 or 20 days separated multiple blackout treatments (e.g. Test 4, Mean = 0.74 ± 0.007 (SE), and Mean = 0.74 ± 0.005). PEF values were significantly different between blackout start dates (P=0.03) and between one and two blackout treatments (P<0.01) on the first measurement date only (October 17), when data were pooled for 10 and 20 days between blackouts in Test 4 (Fig. 21). For this measurement date, values were below the threshold of 0.65 for all three of the blackout start dates, but were higher for the early and middle start dates. Both one and two blackout treatments resulted in PEF values below the threshold on the October 17 measurement date (Fig. 21). PEF for the three start dates (July 12 LSMean = 0.81 + 0.029 (SE), July 26 LSMean = 0.81 ± 0.029 (SE), August 10 LSMean = 0.82 ± 0.030 (SE)), and one or two blackout treatments (LSMean = 0.81 ± 0.029 (SE) and LSMean = 0.81 ± 0.030 (SE), respectively) were similar and high for all other measurement dates pooled.  103  10-Aug Blackout Start Date  Figure 21. Mean photosynthetic efficiency (Fv/Fm) after -18°C freezing (PEF) on the October 12 measurement date with three blackout start dates of July 12, July 26, and August 10, and one or two blackout treatments. Data pooled for 10 and 20 days between multiple blackout treatments. For the normal blackout regime, PEF for the Washington provenance was significantly higher than PEF of the Vancouver Island provenance on the first and last measurement dates before cold storage in Test 5 with Pelton Reforestation's common blackout regime (Fig. 22). PEF for the Vancouver Island provenance was below the threshold on the first measurement date.  104  11  0.3 0.2 J 1-Oct  ,  ,  ,  20-Nov  9-Jan  28-Feb  Measurement Date  Figure 22. Mean ± SE photosynthetic efficiency (Fv/Fm) after -18°C freezing (PEF) for Washington and Vancouver Island provenances. Provenances compared for Pelton Reforestation Limited normal blackout regime. The threshold line indicates an adequate PEF for seedlings to be put into cold storage. On March 16, PEF of those seedlings not cold stored was significantly lower than seedlings cold stored for one month in Test 6, and was below the quality threshold of 0.65 when the data was pooled for the two blackout treatments subjected to cold storage (Table 15). While PEF significantly decreased from February 9 to March 16 with no cold storage, PEF values were similar when seedlings were cold stored between these dates.  Days to terminal bud break (DBB) For the first four measurement dates, seedlings from all treatments were still in deep rest and therefore did not break bud when planted in the greenhouse. For these dates, the shortest period that the seedlings were left in the greenhouse was 47 days, at which time no seedlings were showing signs of terminal bud flush. Therefore, the results for DBB  105  will only be discussed for the January 4, January 24, and February 9 lift dates before cold storage, and the March 16 planting date after cold storage. Terminal bud flush on the January 4 lift date was highly variable and had the highest proportion of seedlings without terminal bud flush by April 17 (20 out of 280 seedlings). Excluding the very few that died, only one seedling from the January 24 lift date did not flush by April 17, and all seedlings planted on February 9 and March 16 flushed by this time.  In all tests on the last three measurement dates dormancy was not affected by blackout start date (e.g. Table A-21, Appendix 5). DBB was also similar whether 10 or 20 days between blackout treatments were used, in all tests (e.g. Test 4, LSMean = 39.12 + 0.44 (SE) and LSMean = 38.86 ± 0.44 (SE), respectively). On the January 4 lift date, treatment with only one blackout resulted in a significantly higher DBB than treatment with two or three blackouts in Test 3 when the data was pooled for July 12 and July 26 blackout start dates (Fig. 23). This trend was also found on the January 24 lift date, although differences between the treatments were substantially decreased. For the February 9 lift date, DBB with one or two blackout treatments were similar, while DBB with one blackout treatment was still significantly higher than that with three blackout treatments. DBB for all blackout treatments in Test 3 significantly decreased with the later lift dates.  106  Figure 23. Least squares mean ± SE days to terminal bud break (DBB) on January 4, January 24, and February 9, 2001, with one, two, or three blackout treatments, and 10 days in between multiple blackouts. Data pooled for blackout start dates of July 12, July 26, and Aug 10, 2000. The Washington provenance was significantly more dormant than the Vancouver Island provenance on January 4 in Test 5. However, dormancy was similar between the two provenances for the January 24 and February 9 measurement dates (Fig. 24).  107  60  -I  55 50 -  • Washington provenance E Vancouver Island provenance  4-Jan  24-Jan  9-Feb  Measurement date  Figure 24. Least squares mean ± SE days to terminal bud break (DBB) on January 4, January 24, and February 9, 2001 for Washington and Vancouver Island provenances subjected to Pelton Reforestation Ltd. normal blackout regime. There was a significant drop in DBB from February 9 to March 16 after 4 weeks cold storage and for non-stored seedlings in Test 6 when the data was pooled for the two treatments receiving cold storage (Table 15). However, DBB was significantly lower on March 16 for seedlings that were not cold stored than for cold stored seedlings (Table 15).  108  4.4 DISCUSSION  4.4.1 Height growth  When blackout began on July 12, the height of the seedlings did not exceed 25 cm by the middle of October (Fig. 16). Maximum height by this time increased with the later blackout start dates. A blackout treatment that starts early in the summer can be used successfully to limit height growth in order to meet target height specifications provided height growth during lag time is taken into account (D'Aoust and Cameron 1986; Eastham 1991).  4.4.2 Caliper  Multiple blackout treatments are commonly used to prevent lammas growth after budset. Lammas growth can cause problems at the nursery because of a delay in the development of dormancy and CH development. Multiple blackouts are also thought to maintain a better balance between height and caliper, which is important to the nursery because their clients generally prefer sturdy seedlings with large caliper (Thompson 1985). In this study, however, multiple blackout treatments did not have this effect. While having little effect on height growth, there was a decrease in caliper growth as the number of blackouts increased (Fig. 17). This may be because caliper growth is more responsive to the fewer hours of active photosynthesis that occurs for seedlings receiving blackout. Also, if heat from the high summer temperatures or respiring seedlings was trapped under  109  the large black curtains surrounding the seedlings receiving blackout, the temperature seedlings receiving blackout are exposed to would be higher. This would cause an increase in respiration for seedlings in blackout and may lead to lower growth. Other researchers have also found that longer duration blackout treatments (3-6 weeks) result in reduced caliper (Eastham 1991; Hawkins and Draper 1991). Although blackout durations were kept constant at 12 days for this study, the multiple blackout treatments may have had a similar effect on caliper as did the long duration blackouts given in the two studies mentioned above.  4.4.3 Root growth capacity (RGC)  The RGC trend seen throughout the winter with the blackout study is typical of the annual cycle of this measure (Ritchie 1985; Ritchie 1990). RGC was lowest in late November, when seedlings were in rest (Fig. 19). The peak in RGC for all measurement dates tested did not occur until February 9, when shoots were still inactive but dormancy had weakened. The peak in RGC for non-cold stored seedlings in Phase 1 of the study occurred on January 14 for the Oregon provenance and on February 4 for the Vancouver Island provenance. The later peak seen with the blackout study may be due to the stocktype or provenance differences between the two study phases, or due to climate variation between the two years.  RGC was not affected by the number of blackout treatments or the number of days between blackout treatments. Therefore, hypotheses lb and lc; "RGC is decreased  110  by...increasing the number of blackout treatments, and increasing the number of days in between multiple blackout treatments", can be rejected.  Hypothesis la, "RGC is decreased by advancing blackout start date", cannot be rejected because blackout start dates of July 12 and July 26 reduced RGC values throughout the fall and early winter compared to the later (August 10) start date (Fig. 20). Eastham (1991) also found this to be the case in her study of Sitka x white spruce hybrids. While the results from Phase 1 indicated that all lift dates, cold storage lengths, and cold storage temperature treatments produced seedlings with an adequate RGC, Phase 1 did not consider the blackout regime given to the Oregon and Vancouver Island provenances before they were lifted.  4.4.4 Photosynthetic efficiency after -18°C freezing (PEF)  In the following discussion it is assumed that CF as a measure of photosynthetic efficiency can be used as an indirect measure of cold hardiness (Burr et al. 2001). The discussion of PEF will relate the results obtained from this measure to other research on CH. Coastal Douglas-fir CH should just begin developing in mid-October and be substantially decreased by the middle of March (Burr 1990). It is therefore not surprising that PEF values below the threshold on the first measurement date of October 17 (Fig. 21) and the last measurement date of March 16 with no cold storage were observed (Table 15). PEF was significantly affected by blackout start date and the number of blackout treatments. Therefore, hypotheses 2a and 2b "PEF is affected by...advancing  111  blackout start date and increasing the number of blackout treatments cannot be rejected. PEF was similar with 10 or 20 days between blackout treatments, allowing hypothesis 2c "PEF is affected by...increasing the number of days between multiple blackout treatments", to be rejected.  Altering the blackout regime only affected PEF on October 17 (Fig. 21). On this date, PEF was lower with one blackout than with multiple blackout treatments. Both one and two blackout treatments had unacceptably low cold hardiness values for all three blackout start dates. Only the Vancouver Island provenance had an unacceptably low cold hardiness on this lift date with the normal blackout regime (Fig. 22). By the November 7 test date, PEF was similar between all blackout treatments, and between Washington and Vancouver Island provenances. Cold storage of coastal Douglas-fir is not conducted before November 10 at British Columbia nurseries (Ministry of Forests 1998). This would suggest that any blackout treatment used in this study would provide sufficient PEF for seedlings from a Washington provenance to be put into cold storage at this date.  PEF for the Washington provenance seemed to be more sensitive to the normal blackout regime than did PEF of the Vancouver Island provenance. PEF was attained more quickly and maintained longer in the Washington provenance than in the Vancouver Island provenance (Fig. 22). Normally, southern latitude provenances develop CH later and lose CH earlier than do provenances from further north because the chance of early spring frosts in northern climates is higher (Koslowski and Pallardy 1997; Hawkins and  112  Shewan 2000; Repo et al. 2000). Acceptable values were already obtained by the first measurement date for the Washington provenance, while the values for the Vancouver Island provenance were still below the threshold. Blackout also caused Washington seedlings to maintain PEF values on February 9 similar to those observed in late fall and early winter, while PEF for the Vancouver Island provenance had been declining since late November. This may indicate that southern provenances are more sensitive to an artificially shortened photoperiod.  CH is initiated by growth cessation (Campbell and Sugano 1975; Christersson 1978; Valkonen et al. 1990; Grossnickle 2000), and earlier CH development would have occurred with those seedlings that set bud early. Blackout allowed for growth cessation to occur earlier than under natural conditions, and likely affected the timing of cold hardiness development. Decreasing temperatures in the fall may have increased the speed of PEF development for those seedlings that had already set bud (Grossnickle 2000).  4.4.5 Days to terminal bud break (DBB)  Maximum dormancy is usually attained in Douglas-fir by October or November (Lavender 1985). Both provenances were already in rest by the first measurement date of October 12. In Douglas-fir, rest is usually broken by mid-February (Lavender 1981). Maximum CH corresponds to the time when the seedling is shifting from rest to postdormancy quiescence (Glerum 1976), suggesting a lag time between the two processes.  113  In this study, a lag between dormancy release and loss of PEF was observed for both Washington and Vancouver Island provenances. On October 12, bud flush would not occur under optimal greenhouse conditions. This indicates that the seedlings were in rest. Cold hardiness was still increasing at this time.  Excluding measurement dates before January 4 (for which bud break did not occur after 45 days), DBB for the last three measurement dates, unlike RGC, was similar between the three blackout start dates. DBB values were unaffected by whether the blackout treatments were separated by 10 or 20 days. That is, blackout start date and the days in between blackout treatments did not affect the speed of dormancy release, and hypotheses 3a, "Dormancy is released more quickly by...advancing blackout start date", and 3c, "Dormancy is released more quickly by...increasing the number of days in between multiple blackout treatments", can be rejected. Eastham (1991) also did not find start date to affect subsequent DBB for sitka x white spruce hybrid seedlings.  Advanced bud break in the field can cause problems for seedlings planted in late winter/early spring. Cold soils and exposure to cool temperatures that are possible at this time of year in many regions of the Pacific Northwest may lead to plant injury (Hawkins and Hooge 1988; Hawkins et al. 1996). Although cool temperatures in Oregon are less likely on lower elevations sites in late winter/early spring, frost damage is still possible, even in southern portions of Oregon (Tesch and Helms 1992). Planting of more dormant seedlings will postpone bud break, causing a delay in shoot growth to when field  114  conditions are more optimal. It will also allow greater root growth before bud break occurs.  Hypotheses 3b, "Dormancy is released more quickly by...increasing the number of blackout treatments", cannot be rejected because dormancy release in January and early February was affected by the number of blackout treatments given in the previous summer (Fig. 23). By early February, however, this difference had substantially decreased. In Phase 1, the Oregon provenance was given two blackout treatments while the Vancouver Island provenance was only subjected to one blackout. This may have contributed to the generally lower dormancy of the Oregon provenance than the Vancouver Island provenance in the following winter and spring (Fig. 24).  4.4.6 Cold storage  RGC, DBB, and PEF significantly decreased from February 9 to March 16 when seedlings were not cold stored (Table 15). However, cold storage maintained RGC and PEF at levels similar to those before storage. This corresponds well with the first phase of this study, in which cold storage was not found to decrease RGC, and with previous research findings (Ritchie 1982; Mattsson and Troeng 1986; Dunsworth 1988). A higher value for RGC, DBB and PEF was noted for the cold stored stock as compared to nonstored stock on March 16, which is also consistent with the results from Phase 1 of this study that cold storage delays dormancy release. A cold storage temperature of -2°C is not optimal for meeting the chilling requirement of Douglas-fir seedlings (Ritchie 1984;  115  Ritchie et al. 1985; Ritchie 1987). If the seedlings had not met their chilling requirement by the lift date for cold storage (February 9), it may have been fulfilled more slowly in storage.  4.4.7 Relating study results to 1999 and 2000 field observations in Oregon  The 1999 and 2000 Pelton stock produced for Oregon clients that exhibited poor field performance were planted in early February without cold storage. A planting date at this time is within the window (February - May) suggested for planting Douglas-fir in Oregon (Randall and Johnson 1998). Seedlings from the first phase were from the same stock as those that failed after planting in Oregon in 2000. Phase 1 results indicated that RGC and DDBB were low for Oregon provenance seedlings not cold stored by early February. However, the seedlings tested in Phase 2 had high RGC and PEF in early February. Differences between the two phases may have been due to climatic variation between 2000 and 2001, genetic differences between the Washington and Oregon provenances, or because of differences in stocktype.  Differences in RGC, DBB, and PEF were found between blackout treatments in Phase 2 of this study. However, these differences were either small, did not result in values below the quality thresholds, or occurred before the spring planting window. In Phase 1, only thawing period resulted in RGC and CF values below the threshold. Study site conditions at the Malcom Knapp research forest were not stressful enough to notice large treatment differences in the field performance study. The differences noted in Phase 1  116  and Phase 2 of this study may have been more pronounced had the seedlings been exposed to stressful conditions mimicking the natural Oregon field site conditions in 1999 and 2000. It is also likely that field performance in Phase 1 may have correlated better with RGC and DDBB had the seedlings been planted under more stressful field conditions.  117  5.0 CONCLUSION  The range of treatments used in Phase 1 was chosen to incorporate the variety of nursery handling techniques used at Pelton Reforestation and other container nurseries in the Pacific Northwest for coastal Douglas-fir. Initial survival potential of Douglas-fir from Oregon and Vancouver Island provenances is not decreased below an acceptable threshold with the range of lift dates, cold storage lengths, and cold storage temperatures commonly used by seedling nurseries, provided a one-week thawing period is used with minimal on-site storage. Extending the thawing period or on-site storage can result in lower initial survival potential (RGC and CF), particularly after longer cold storage. Later lift dates, longer storage lengths and longer thawing periods resulted in faster dormancy release. This can be important in spring planting, since less dormant seedlings generally have lower resistance to stresses such as drought and cold temperatures.  Although correlations were found between the measures of initial survival potential and dormancy, no one measure can be relied on as an indicator of overall seedling quality. This was indicated by the lack of a strong correlation between measures of initial survival potential and dormancy, and the field performance measures. Stronger correlations may have been found had field site conditions been more stressful.  Trees from all Phase 1 treatments grew well in the field performance study at the Malcom Knapp research forest, including those from treatments with a relatively low initial survival potential. Survival of trees from all treatments was similar and high at  118  approximately 90%. This may indicate that the threshold levels used for the initial survival potential measures in Phase 1 of this study were too conservative and could be lowered for more accurate predictions. However, treatment differences may have been more evident had the field site conditions been more stressful. The Oregon provenance exhibited a lower RGC and faster dormancy release before planting, and a higher incidence of multiple leaders in the field compared to the Vancouver Island provenance, which is consistent with problems observed in Oregon in 1999 and 2000. Provenance differences were investigated in Phase 2 of this study to determine if they were influenced by blackout regime.  Phase 2 was conducted to determine how blackout affected seedling phenology and morphology. One limitation was the inability to use the same provenances for both phases. Phase 2 used a Washington provenance and a Vancouver Island provenance as compared to Phase 1, which used an Oregon provenance and a different Vancouver Island provenance. The earliest blackout start date resulted in the lowest RGC. While it is unlikely that the root growth problems seen in Oregon in 1999 and 2000 were caused by lift/storage conditions tested in Phase 1, it is possible that an early blackout start date contributed to the lower root growth of the Oregon provenance in the early spring. The high RGC values seen in the fall for all blackout start dates indicate that Douglas-fir stock could be planted in the early fall, especially in Washington and Oregon where climate conditions can still be favourable at this time.  119  Dormancy was released more quickly with multiple blackout treatments. PEF was higher in the early fall for seedlings treated with multiple blackouts. However, this trend disappeared by November 23. Cold storage and/or a less intensive blackout regime resulted in higher RGC, higher cold hardiness, and a slower dormancy release after planting.  120  6.0 RECOMMENDATIONS  1. Careful attention should be paid to the thawing regime conducted at both the nursery and by the seedling user. A rapid thaw at high temperatures (10°C) or a slower thaw at lower temperatures (1-2°C) is likely to improve seedling performance after outplanting. 2. On-site storage should be avoided, especially if seedlings have thawed for longer than one week. 3. Hot-lifted Oregon provenances that have received blackout should be planted in early October, when stock is dormant, and PEF and RGC are adequate. If seedlings must be planted in the spring, they should be put into cold storage in early January so that high RGC, DBB, and PEF can be maintained. 4. Two possibilities for further study would be: a) to investigate the link between early dormancy release and poor root growth for container seedlings planted in early spring and, b) a comparison of the effects of blackout versus drought stress dormancy induction techniques on seedling phenology, morphology, and physiology. c) further investigation into the thawing period for Douglas-fir at a range of temperatures to determine the maximum number of days for thawing before seedling quality declines.  121  7.0 LITERATURE CITED Aitken, S.N. and W.T. Adams. 1997. Spring cold hardiness under strong genetic control in Oregon populations of Pseutosuga menziesii var. menziesii. Can. J. For. Res. 27:1773-1780. Arnott, J.T. and A. Mitchell. 1981. Influence of extended photoperiod on growth of white spruce and engelmann spruce seedlings in coastal British Columbia nurseries. In Proc. of the Canadian Containerized Tree Seedling Symposium. J.B. Scarratt, C. Glerum, and C A . Plexman (editors). Canada-Ontario Joint Forest Research Commission, Toronto, Ont. Proc. O-P-10. pp. 139-152. Binder, W.D., R.K. Scagel, and G.J. Krumlik. 1988. Root growth potential: facts, myths, value? In Proc. of the 1988 combined meeting of the Western Forest Nursery Associations. T.D. Landis (Tech. Coordinator). Vernon, B.C. General Technical Report RM-167. pp. 111-118. Binder, W.D., P Fielder, R. Skagel, and G.J. Krumlik. 1990. Temperature and timerelated variation of root growth in some conifer tree species. Can. J. For. Res. 20:1192-1199. Burr, K.E. 1990. The target seedling concept: bud dormancy and cold-hardiness. In Target Seedling Symposium: Proc. of a Combined Meeting of the Western Forest Nursery Associations. USDA Forest Service. General Technical Report RM200. pp. 79-90. Burr, K.E. and R.W. Tinus. 1988. Effect of the timing of cold storage on cold hardiness and root growth potential of Douglas-fir. General Technical Report R M 167. pp. 133-138. Burr, K.E., R.W. Tinus, S.J. Wallner and R.M. King. 1989. Relationships among cold hardiness, root growth potential and bud dormancy in three conifers. Tree Physiol. 5:291-306. Burdett, A.N. 1979. New methods for measuring root growth capacity: their value in assessing lodgepole pine stock quality. Can. J. For. Res. 9:63-67. Camm, E.L., D.C. Goetze, S.N. Silim, and D.P. Lavender. 1994. Cold storage of conifer seedlings: an update from the British Columbia perspective. For. Chron. 122  70:311-316 Camm, E.L., R.D. Guy, D.S. Kubien, D.C. Goetze, S.N. Silim, and P.J. Burton. 1995. Physiological recovery of freezer-stored white and Engelmann spruce seedlings planted following different thawing regimes. New For. 10:55-77 Campbell, R.K. and F.C. Sorensen. 1973. Cold-acclimation in seedling Douglas-fir related to phenology and provenance. Ecology 54:1148-1151. Campbell, R.K. and A.I. Sugano. 1975. Phenology of bud burst in Douglas-fir related to provenance, photoperiod, chilling, and flushing temperature. Bot. Gaz. 136:290298. Campbell, R.K. and A.L Sugano. 1979. Genecology of bud-burst phenology in Dougalsfir: response to flushing temperature and chilling. Bot. Gaz. 140:223-231. Cannell, M.G.R., P.M. Tabbush, J.D. Deans, M.K. Hollingsworth, L.J. Sheppard, J.J. Philipson, and M.B. Murray. 1990. Sitka spruce and Douglas-fir seedlings in the nursery and in cold storage: root growth potential, carbohydrate content, dormancy, frost hardiness and mitotic index. Forestry 63:9-27. Carlson, W.C. 1978. The use of periodic moisture stress to induce vegetative bud set in Douglas-fir seedlings. In Proc. Int. Plant Prop. Soc, Oct. 3-4, 1978. No. 28. pp. 49-58. Christersson, L. 1978. The influence of photoperiod and temperature on the development of frost hardiness in seedlings of Pinus silvestris and Picea abies. Physiol. Plant. 44:288-294. Chomba, B. 1992. 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Survival of coastal and interior Douglas-fir seedlings after storage at different temperatures, and effectiveness of cold storage in satisfying chilling requirements. Can. J. For. Res. 7:125-131. 1987. Importance of current photosynthate to new root growth in planted conifer seedlings. C. J. For. Res. 17:776-782. 1991a. New root growth of Douglas-fir seedlings at low carbon dioxide concentration. Tree physiol. 8:289-295. 1991b. Influence of container nursery regimes on drought resistance of seedlings following planting. I. Survival and growth. Can. J. For. Res. 21:555-565. Van Eerden, E. and J.W. Gates. 1990. Seedling production and processing: Container. In Regenerating British Columbia's Forests. D.P. Lavender, R. Parish, C. Johnson, G. Montgomery, A. Vyse, R.A. Willes, and D. Winston (editors).  132  University of British Columbia Press, Vancouver, B.C. pp. 226-234. Wareing, P.F. 1985. Plant cell responses and the role of growth substances. Pp 1-9. In Plant growth substances 1985: Proc. of the 12 International Conference on Plant th  Growth Substances. Martin Bopp (editor). Spring-Verlag, Berlin. Weiser, C J . 1970. Cold resistance and injury in woody plants. Science 169:1269-1278. Williams, H.M., D.B. South, and G.R. Glover. 1988. Effects of bud status and seedling biomass on root growth potential of lobolly pine. Can. J. For. Res. 18:1635-1640. Cited in Grossnickle 2000. Wommack, D.E. 1964. Temperature effects on the growth of Douglas-fir seedlings. Ph.D. Dissertation, Oregon State University, Corvallis, OR. 176 pp. Zwiazek, J.J., S. Renault, C. Croser, J. Hansen, and E. Beck. 2001. Biochemical and biophysical changes in relation to cold hardiness. In Conifer Cold Hardiness. F.J. Bigras and S.J. Colombo (editors). Kluwer Academic Publishers,  133  APPENDICES  APPENDIX I Table A - l . Example of General Linear Model and statistical values for RGC in Phase la. Data includes four lift dates, two cold storage lengths, two provenances, and two thawing periods. Source  DF  Sum of Squares  F Value  Pr>F  Provenance (P) Lift (L) Storage (S) Extended Thaw (E) P*L P*S P*E L*S L*E S*E P*L*S P*S*E L*S*E Error Corrected Total  1 3 1 1 3 1 1 3 3 1 3 1 3 610 635  54043.1 43895.9 3711.2 230763.5 1097.4 927.9 2531.3 32649.9 25388.8 37.2 13707.9 5614.7 64618.0 707795.4 1182972.1  46.58 12.61 3.20 198.88 0.32 0.80 2.18 9.38 7.29 0.03 3.94 4.84 18.56  0.0001 0.0001 0.0742 0.0001 0.8144 0.3716 0.1402 0.0001 0.0001 0.8579 0.0084 0.0282 0.0001  Table A-2. Example of General Linear Model and statistical values for Caliper increment in Phase lb. Data includes four lift dates, two cold storage lengths, and two provenances. Error split into sampling and experimental errors because 50 seedlings from each treatments were planted in rows of five. Source  DF  Sum of Squares  F Value  Pr>F  Storage length (S) Lift (L) Provenance (P) L*S S*P L*P L*S*P Sampling error Experimental error Corrected Total  1 3 1 3 1 3 3 16 761 792  0.1 6.6 46.2 8.0 40.5 8.6 40.3 128.8 1493.6 1789.7  0.01 0.27 5.74 0.33 5.03 0.35 1.67  0.9149 0.8445 0.0292 0.8034 0.0394 0.7866 0.2139  135  Table A-3. Example of General Linear Model and statistical values for RGC in Phase 2 study. Data includes seven test dates, three blackout start dates, one and two blackout treatments, and 10 and 20 days between blackouts. Error split into sampling and experimental errors because 20 seedlings from each treatments were planted in rows of four in the UBC agricultural greenhouse. Source  DF  Sum of Squares  F Value  Pr>F  Test Date (T) Start date (S) # of blackouts (B) Days between blackouts (D) T*B T*D S*B T*S T*B*D T*S*B S*B*D S*B*D T*S*B*D Sampling error Experimental error  6 2 1 1  80.4 7.0 0.3 0.1  11.35 2.96 0.22 0.10  0.0001 0.0570 0.6423 0.7542  6 6 2 12 6 12 12 2 12 84 150  10.2 3.7 1.2 11.5 3.7 13.1 4.0 0.4 4.0 722.7 1271.3  1.44 0.52 0.50 0.82 0.52 0.92 0.28 0.17 0.28  0.2086 0.7914 0.6093 0.6363 0.7914 0.5273 0.9904 0.8445 0.9904  Corrected error  e  o 167 5  99.2  136  APPENDIX II  16  T  .41 Julian Date  Figure A - l . Daily minimum temperature from January 1 to Oct 31 measured at the meteorological station in Malcom Knapp Research Forest.  50 j 45  -i  40 -  0  50  100  150  200  250  300  Julian Date  Figure A-2. Daily precipitation from January 1 to Oct 31, 2000 measured at the meteorological station in Malcom Knapp Research Forest.  137  APPENDIX III Table A-4. Summary of Oregon provenance seedling quality reported by seedling users for Pelton Reforestation Limited stock. Company  Year  Method of Observation  Observation  Weyerhauser Roseburg, OR.  1999  Field observations  2000  Field observations  Roseburg Forest Products Roseburg, OR.  2000  RGC tests (Lebanon Forest RGC below average Regeneration Center)  Swanson Superior Eugene, OR.  2000  Field observations  1) Poor root growth 2) Early and vigorous shoot growth Good shoot and root growth  1) Poor root growth 2) Terminal shoot damage  138  Figure A-3. Poor root development of Douglas-fir seedlings planted in February 2000, and produced at Pelton Reforestation (615D stocktype). Removed from Swanson Superior Forest Products field site near Noti, Oregon in July 2000.  Figure A-4. Poor terminal shoot development of Douglas-fir seedlings planted in February 2000, and produced at Pelton Reforestation (615D stocktype). Removed from Swanson Superior Forest Products field site near Noti, Oregon in July, 2000.  APPENDIX IV Table A-5. Probability values associated with Test 1 in Phase la for root growth capacity (RGC), chlorophyll fluorescence (CF), and degree-days to terminal bud break (DDBB). RGC Provenance (P) 0.001 Lift date (L) 0.001 Storage duration (S) 0.11 P*S 0.19 P*L 0.005 L*S 0.001 P*L*S 0.001  CF 0.38 0.001 0.001 0.003 0.002 0.001 0.001  DDBB 0.001 0.001 0.001 0.001 0.001 0.001 0.24  Table A-6. Probability values or significant interactions associated with Test 2 in Phase la for root growth capacity (RGC), chlorophyll fluorescence (CF), and degree-days to terminal bud break (DDBB). Provenance (P) Lift date (L) Storage duration (S) Thawing period (Tp) Significant Interactions (P<0.03)  RGC 0.001 0.001 0.07 0.001 L*S, L*Tp, P*L*S, P*S*Tp, L*S*Tp  CF 0.001 0.001 0.001 0.001 P*L, P*S, P*Tp, L*S, L*Tp, S*Tp, P*S*Tp, L*S*Tp  DDBB 0.18 0.001 0.001 0.001 P*L, P*S, P*Tp, L*S, L*Tp, S*Tp, L*S*Tp  Table A-7. Probability values associated with Test 3 in Phase la for root growth capacity (RGC), chlorophyll fluorescence (CF), and degree-days to terminal bud break (DDBB). Storage duration (S) Temp (T) Provenance (P) S*T T*p S*P S* [ *p r ,  RGC 0.001 0.001 0.08 0.001 0.004 0.33 0.003  CF 0.03 0.002 0.05 0.57 0.15 0.06 0.007  DDBB 0.001 0.81 0.003 0.16 0.09 0.02 0.77  140  Table A-8. Probability values associated with Test 1 in Phase lb for relative height, relative caliper, frequency of multiple leaders (FML), and frequency of chlorotic seedlings (FCS). Relative height Storage (S) 0.19 Lift (L) 0.76 Provenance (P) 0.001 Thawing period 0.001 (Tp) none Significant Interactions  Relative caliper FML 0.15 0.60 0.78 0.57 0.24 0.39 0.24 0.14 S*P  P*E  FCS 0.25 0.13 0.95 0.05 none  Table A-9. Probability values associated with Test 2 in Phase lb for relative height, relative caliper, frequency of multiple leaders (FML), and frequency of chlorotic seedlings (FCS).  Storage (S) Lift (L) Provenance (P) L*S L*P P*S L*S*P  Relative height 0.55 0.59 0.12 0.71 0.91 0.09 0.96  Relative caliper FML 0.31 0.10 0.48 0.68 0.51 0.16 0.27 0.11 0.46 0.71 0.08 0.36 0.80 0.19  FCS 0.22 0.33 0.12 0.47 0.68 0.17 0.14  Table A-10. Probability values associated with Test 3 in Phase lb for relative height, relative caliper, frequency of multiple leaders (FML), and frequency of chlorotic seedlings (FCS).  Storage (S) Lift (L) Provenance (P) L*S L*P P*S L*S*P  Relative height 0.35 0.49 0.14 0.98 0.64 0.37 0.28  Relative caliper FML 0.94 0.26 0.67 0.42 0.61 0.44 0.32 0.28 0.06 0.78 0.86 0.35 0.39 0.83  FCS 0.95 0.37 0.16 0.22 0.62 0.40 0.38  141  Table A-11. Probability values associated with Test 4 in Phase lb for relative height, relative caliper, frequency of multiple leaders (FML), and frequency of chlorotic seedlings (FCS).  Storage (S) Lift (L) Provenance (P) L*S L*P P*S L*S*P  Relative height 0.52 0.56 0.03 0.99 0.92 0.02 0.48  Relative caliper FML 0.88 0.66 0.86 0.38 0.17 0.70 0.54 0.85 0.80 0.15 0.02 0.50 0.19 0.19  FCS 0.97 0.23 0.66 0.94 0.90 0.85 0.60  Table A-12. Probability values associated with Test 5 in Phase lb for relative height, relative caliper, frequency of multiple leaders (FML), and frequency of chlorotic seedlings (FCS). Thawing period (Tp) Lift (L) Provenance (P) Tp*L L*P Tp*P Tp*L*P  Relative height 0.02  Relative caliper FML 0.90 0.48  FCS 0.36  0.92 0.01 0.56 0.93 0.42 0.40  0.78 0.64 0.62 0.62 0.08 0.80  0.42 0.29 0.52 0.60 0.81 0.49  0.33 0.45 0.27 0.25 0.76 0.38  Table A-13. Probability values associated with Test 6 in Phase lb for relative height, relative caliper, frequency of multiple leaders (FML), and frequency of chlorotic seedlings (FCS). Temperature Provenance Storage P*S T*S  Relative height 0.16 0.09 0.26 0.64 0.64 0.62 0.65  Relative caliper FML 0.52 0.24 0.22 0.27 0.41 0.79 0.47 0.91 0.35 0.79 0.18 0.36 0.77 0.36  FCS 0.24 0.93 0.24 0.47 0.30 0.68 0.30  142  Table A-14. Probability values associated with Test 1 in Phase 2 for root growth capacity (RGC), photosynthetic efficiency after -18°C freezing (PEF), and days to terminal bud break (DBB).  Blackout number (B) Measurement date (M) B*M  RGC 0.16 0.001  PEF 0.08 0.001  DBB 0.10 0.001  0.08  0.86  0.11  Table A-15. Probability values associated with Test 2 in Phase 2 for root growth capacity (RGC), photosynthetic efficiency after -18°C freezing (PEF), and days to terminal bud break (DBB).  Blackout number (B) Days between blackouts (D) Measurement date (M) B*M B*D D*M D*B*M  RGC 0.92 0.46  PEF 0.001 0.07  DBB 0.003 0.27  0.003  0.001  0.001  0.78 0.84 0.49 0.90  0.006 0.16 0.08 0.15  0.02 0.20 0.86 0.91  Table A-16. Probability values associated with Test 3 in Phase 2 for root growth capacity (RGC), photosynthetic efficiency after -18°C freezing (PEF), and days to terminal bud break (DBB).  Start date (S) Blackout number (B) Measurement date (M) B*M B*S S*M S*B*M  RGC 0.07 0.99 0.001  PEF 0.79 0.01 0.001  DBB 0.37 0.69 0.001  0.85 0.63 0.72 0.75  0.009 0.46 0.18 0.01  0.87 0.43 0.61 0.32  Table A-17. Probability values associated with Test 4 in Phase 2 for root growth capacity (RGC), photosynthetic efficiency after -18°C freezing (PEF), and days to terminal bud break (DBB).  Start date (S) Blackout number (B) Days between blackouts (D) Measurement date (M) Significant interactions (P<0.05)  RGC 0.056 0.64 0.75  PEF 0.37 0.01 0.94  DBB 0.94 0.92 0.90  0.001  0.001  0.001  none  S*B*M  none  Table A-18. Probability values associated with Test 5 in Phase 2 for root growth capacity (RGC), photosynthetic efficiency after -18°C freezing (PEF), and days to terminal bud break (DBB).  Provenance (P) Measurement date (M) P*M  RGC 0.32 0.001  PEF 0.001 0.001  DBB 0.06 0.001  0.43  0.003  0.32  Table A-19. Probability values associated with multiple comparisons analysis in Phase 2 (Test 6) for root growth capacity (RGC), photosynthetic efficiency after -18°C freezing (PEF), and days to terminal bud break (DBB).  Before cold storage: RGC PEF DBB No cold storage: RGC PEF DBB  After cold storage  No cold storage  0.25 0.82 0.001  0.003 0.001 0.001  0.03 0.001 0.003  144  APPENDIX V Table A-20. Least squares mean (±SE) values (relative height increment and relative caliper increment) and mean values (frequency of multiple leaders and frequency of chlorotic seedlings) for similar results with the provenance*temperature interaction in Phase lb (Test 6). Data pooled for 4 and 10 weeks cold storage. Measure  Provenance  Temperature  LSMean Value (±SE)  Relative height increment  Oregon  -2°C +1°C -2°C +1°C -2°C +1°C -2°C +1°C  0.69(±0.049) 0.70(±0.050) 0.71 (±0.050) 0.63(±0.050) 0.58(±0.049) 0.56(±0.049) 0.52(±0.049) 0.50(±0.049)  Vancouver Is. Relative caliper increment  Oregon Vancouver Is.  Measure  Provenance  Temperature  Mean Value (±SE)  Frequency of multiple leaders  Oregon  -2°C +1°C -2°C +1°C -2°C +1°C -2°C +1°C  0.07(±0.026) 0.07(±0.026) 0.05(±0.020) 0.06(±0.024) 0.07(±0.017) 0.10(±0.030) 0.11 (±0.031) 0.10(±0.030)  Vancouver Is. Frequency of chlorotic seedlings  Oregon Vancouver Is.  Table A-21. Least squares mean (±SE) RGC values with a varying number of blackout treatments (Test 1). Number of blackout treatments 1 2 3 4  RGC LSMean (±SE) 3.60(±0.063) 3.50(±0.063) 3.52(±0.063) 3.54(±0.063)  Table A-22. Least squares mean (±SE) DBB values with a varying number of blackout treatments (Test 1). Blackout start date July 12 July 26 Aug. 10  DBB LSMean (±SE) 38.59(±0.54) 39.18(±0.54) 39.20(±0.53)  145  

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