MSU LIBRARIES RETURNING MATERIALS: PIace in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. a d 4"! '1‘“ an ENVIRONMENTAL CONTROL OF SPRUCE SEEDLING GROWTH AND SHOOT DEVELOPMENT BY John William Heckman, Jr. A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Forestry 1985 ACKNOWLEDGEMENT I thank Dr. James W. Hanover (committee chairman) who provided guidance, encouragement, and the opportunity to pursue a doctoral degree at Michigan State University; Drs. K. K. Baker, S. K. Ries, and D. A. Reicosky who served on my guidance committee; I especially thank Drs. K. K. Baker and EL L. Flegler of the Center for Electron Optics for their excellent instruction in the practice of electron microscopy and the virtually unrestricted use of the CenterHs equipment; my fellow graduate students for their willing and helpful critique; my family for their continued support in this endeavor; and most especially to my wife, Betty Jane whose tireless support and good humor helped sustain me throughout this period. ABSTRACT ENVIRONMENTAL CONTROL OF SPRUCE SEEDLING GROWTH AND SHOOT DEVELOPMENT by John William Heckman, Jr. The photoperiodic protraction of indeterminate (free) growth is important in commercial seedling production. Free growth varies among species and with age. In spruce, the mature growth pattern is determinate. Only young seedlings free grow. The objective of this study was to improve the understanding of seedling development during this growth- pattern change. Free growth dynamics of spruce seedlings were followed for 24 weeks. Relative biomass growth rates were constant: suggesting exponential growth. Height growth was also studied. Relative height growth rates declined rapidly. Apical meristem development was also followed. Measurements were made with a digitizer and a volumetric computer program. Meristem volumes increased up to 10 weeks; cytohistological zone proportions continued to change. The pith-rib meristem relative volume increased at the expense of the others zones. Shortened growth cycles (SGC) were used to determine the effects of cyclic growth on free growth potential. All species could free grow, even after four cycles. SGC seedlings were smaller than normally cycled seedlings. The buds were also smaller than normal. SGCs seemed to impede development. John William Heckman, Jr. Seedling age and size effects on free growth were tested. Blue spruce seedlings of three sizes, grown by "accelerated growth" (AG) and "conventional nursery" (NUR), methods were compared under long days (LD) and short days (SD). Within each size class, AG seedlings were less developed than NUR seedlings. Photoperiod had no effect on flushing. NUR seedlings grew faster and more than AGs, during the flush, but had shorter internodes. NUR seedlings free grew less than AGs, all of which free grew under LD. Some NUR seedlings did not free grow. Reduced growth potential, perhaps due to root disturbance and low light levels, was more important than seedling size. Normal bud development occurred under SD. By the last sample, SD meristems had no cytohistological zonation and cell walls appeared shrunken. SD meristem cells had smaller vacuoles and more starch grains than LD cells. Free growing meristems had distinct zonation and were like active meristems previously described in spruce. Seedlings not free growing under LD had meristems like those normally seen during late bud scale initiation. Prolonged bud scale formation seems to occur in seedlings not free growing under LD. TABLE OF CONTENTS Page LIST OF TABLES OOOOOOOOOOOOOOOOOOOO...OOOOOOOOOOOOOOOOO v LIST OF FIGURES OOOOOOOOOOOCOOOO0.00.00.00.00.000...... Vi INTRODUCTION O0.0.00.0...OOOOOOOOOIOOOOOOOOOOOO00...... 1 CHAPTER 1. The Growth Dynamics of Spruce Seedlings in a Uniform Environment. Abstract ................................... Introduction ............................... Materials and Methods ...................... ReSUItS IO..00...I...OOOOOIOOOOOOOOOOOOOOOOO 1 DiSCUSSion OOOIOOOOOIOIOOOOOOOOOCOOOOOOOOOOO 3 ONOQO‘ 2. The Development of the Spruce Shoot Apical Meristem in a Uniform Environment. Abstract ................................... 34 Introduction ............................... 36 Materials and Methods ...................... 38 Results .................................... 44 Discussion ................................. 62 3. The Effect of Shortened Growth Cycle Number on SpruceeSeedling Apical Development and Free Growth Potential. Abstract ................................... 67 Introduction ............................... 68 Materials and Methods ...................... 72 Results .................................... 74 Discussion ................................. 88 4. The Effects of Seedling Size and Age and Cultural Method on Subsequent Photoperiodically-Mediated Growth in Blue Spruce Seedlings. Abstract ................................... 94 Introduction ............................... 96 Materials and Methods ...................... 98 Results .................................... 101 Discussion ................................. 113 iii CHAPTER Page 5. The Effects Photoperiod on Apical Development, Anatomy, and Ultrastructure in Blue Spruce Seedlings. Abstract ................................... 119 Introduction ............................... 121 Materials and Methods ...................... 123 Results .................................... 126 Discussion ................................. 152 CONCLUSIONS AND RECOMMENDATIONS OOOOOOOOOOOOOCOCOOCOOOO 165 BIBLIOGRAPHY OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 171 Table 1.1 1.3 1.4 4.2 5.1 LIST OF TABLES Selected morphological characteristics of blue, hybrid, white and Norway spruce germinant seedlings after 24 weeks of growth under 24 hr photoperiod . . . . . . . Regression models tested for comparing seedling biomass growth for blue, hybrid, white and Norway spruce, under 24 hr photoperiods for 24 weeks. .. . .. . .. . Regression models tested for comparing seedling height growth for blue, hybrid, white, and Norway spruce, under 24hr photoperiods for 24 weeks. .. . .. . .. . Regression equations predicting plastochron index from height for each species .. .. . Spearman rank correlations between shoot apical meristem developmental characteristics and selected growth indices for pooled spruce apical meristem data .. . . .. . . .. . . Apical meristem volumes and needle primordia seen in spruce buds developed under SGC and in nursery grown and mature buds . .. .. . Selected initial characteristics of commercial blue spruce nursery stock grown under conventional. methods (NUR) for two years (2+0) and under accelerated growth conditions (AG) for one year .. . . .. . . .. . . .. . . Selected characteristics of 2-year-old blue spruce nursery stock (NUR) and one-year-old, accelerated-growth (AG) stock after flushing and growth for 16 weeks under 12 hr (SD) and 24 hr (LD) photoperiods.. . .. . .. . .. Selected initial characteristics of the terminal buds of commercial blue spruce nursery stock grown under conventional methods (NUR) for two years (2+0) and under containerized, accelerated-growth conditions (AG) for one year. 0 O O O O O O O O O O O O O I O O O O O Page 13 19 23 27 61 87 102 112 126 Figure 1.1 1.2 1.3 2.1 2.2 2.3 LIST OF FIGURES Cumulative biomass growth of blue, hybrid, white and Norway spruce seedlings grown for 24 weeks under 24 hr photoperiod. .. .. Weekly mean relative growth rates (RGR) for spruce seedlings grown for 24 weeks under 24 hr phOtoperiOd O O O O O O O O O O O I O 0 Cumulative height growth of blue, hybrid, white and Norway spruce seedlings grown for 24 weeks under 24 hr photoperiod.. . . . Weekly mean relative height growth rates (RHR) for spruce seedlings grown for 24 weeks under 24 hr phOtOperiOd O O O O O O O O O I O O O I Seedling plastochron index (PI) for blue, hybrid, white, and Norway spruce seedlings grown for 24 weeks under 24 hr photoperiod . . . . . . . . Median longitudinal section through the shoot apical meristem of a representative 16-week -old Norway spruce seedling . .. . .. .. . Median longitudinal sections through the apical meristems of a spruce embryo and S-week-old seedling. O O O O O O O O O O O O O O O O 0 Median longitudinal sections through the shoot apical meristems of blue spruce seedlings at different times during their development. . . Median longitudinal sections through the shoot apical meristems of hybrid spruce seedlings at different times during their development . Median longitudinal sections through the shoot apical meristems of white spruce seedlings at different times during their development. . . Median longitudinal sections through the shoot apical meristems of Norway spruce seedlings at different times during their development . vi Page 13 17 21 25 28 41 45 48 50 52 54 Figure 2.7 3.3 3.5 Page Spruce seedling shoot apical meristem volume, radius and height development under 24 hr photoperiod . . . . . . . . . . . . . . . . . 55 Relative volumes of cytohistological zones within the developing shoot apical meristems of blue, hybrid, white and Norway spruce grown for 24 weeks under 24 hr photoperiod. . 57 Mean heights of seedlings of blue, hybrid, white, and Norway spruce at the end of 1 to 5 shortened growth cycles under short days (SD) and their response to long days (LD) . . 76 Average height growth of spruce seedlings under 24 hr photoperiod after 1 to 4 shortened growth cycles.. . .. . .. . .. . . .. . 78 Representative median longitudinal sections through the terminal bud of blue spruce seedlings after 1 to 4 shortened growth cycles . . . . . . . . . . . . . . . . . . . . 81 Representativeemedian longitudinal sections through buds of spruce seedlings after four shortened growth cycles.. . .. . .. . .. . 83 Representative»median longitudinal sections throughnaturally-cycled dormant blue spruce buds.. . .. . .. . . .. . .... . . 85 Representativelilue spruce seedlings of the three size classes and two nursery culture methods, tested for free growth potential. .. 104 New flush heights for AG and NUR seedlings grown for 16 weeks under two photoperiods.. . 106 Biomass accumulation and distribution in NUR and AG blue spruce seedlings grown for 16 weeks under 12 hr and 24 hr photoperiods . . . 109 Preformed shoots from buds of AG and NUR blue sprucee seedlings . .. . . .. . . .. . .. . 128 Light and transmission electron micrographs of the shoot apical meristem of a dormant blue spruce seedling, prior to the start of elongation . . . . . . . . . . . . . . . . . . 130 vii Figure Page 5.3 Average total apical meristem volumes for AG and NUR blue spruce seedlings, pooled over heights, grown under LD and SD photoperiods for 16 weeks. 0 O O O O O O O O O O O O O O O O 135 5.4 Apical regions of AG blue spruce seedlings after flushing and growth under 12 hr and 24 hr photoperiods. . . . . . . . . . . . . . . 138 5.5 Apical regions of NUR blue spruce seedlings after flushing and growth under 12 hr and 24 hr photoperiods. . . . . . . . . . . . . . . 140 5.6 Transmission electron micrographs of cells of the shoot apical meristem of a representative AG seedling grown under SD. .. . .. . .. . . 145 5.7 Transmission electron micrographs of cells of the shoot apical meristem of a representative NUR seedling that showed free growth under LD . 147 5.8 Light and transmission electron micrographs of the shoot apical region of a NUR seedling not resuming free growth under LD photoperiod .. . 150 viii INTRODUCT ION The growth of trees in containers has been studied at least since the investigations of Van Helmont, in 1648 (in Kimball, 1969). Since that time, growing trees in containers has progressed from a botanical curiosity to a major source of seedlings for a variety of silvicultural and horticultural endeavors. It is estimated that over one half billion containerized forest tree seedlings are produced annually, in the northern hemisphere (Hulton, 1974; Reese and Scarratt, 1982). One of the major discoveries facilitating containerized seedling production was that, in the early part of this century, of photoperiodism in forest tree seedlings (see Wareing, 1956 for early review). By combining the optimum climate of a properly regulated greenhouse, a controlled growing medium, and an altered photoperiod, years can be stripped off the the normal nursery period for many tree species (3.3. Hanover e_t a_l_., 1976; Tinus and McDonald, 1979). In order to prudently alter the photoperiodic regimes, it is essential to understand the response of the developing seedling to differing photoperiods. Spruce (Picea s&) is a genus of Northern Hemisphere forest trees generally found in the cooler climatic regions. Several species are commercially important. As a mature tree, spruce exhibits a determinate form of shoot growth. 2 All of the needles and their subtending internodes for a given year are preformed during the preceding summer. They lie dormant, as preformed shoots in the vegetative buds, through the winter. The spruce embryo has no preformed leaves, except the cotyledons. The preformed shoot thus consists only of a primordial apical meristem and the hypocotyl. Therefore, the spruce seedling must change from a completely indeterminate form of growth to the cyclic determinate form mentioned above. This appears to occur within the first few years for spruce grown under natural conditions (Pollard and Logan, 1976; Young and Hanover,1976). It is during this early development, especially in the first year, that the greatest advantages of containerized spruce seedling production are realized. Blue spruce (Picea pungens Engelnu), Norway spruce (g; abies (L.) Karst.), and white spruce (E; 313253 (Moench.) Voss) are species of commercial importance, in northern temperate zones. Greenhouse production of seedlings of these species, using containers and artificially extended photoperiods, is also increasing. The long day (or short night) photoperiodic induction and control of indeterminate or "free" growth in spruce germinant seedlings is well documented and seems to hold for all species (_e_._g_. Hanover e_t a_l_., 1976). Less is known about their growth and developmental dynamics. As more and greater investments are made in containerized seedling production and 3 energy costs increase, a knowledge of how seedlings grow, under various photoperiodic regimes, will have increasing economic importance. The role of photoperiod in the whole of seedling development, in addition to the induction of free growth, is being increasingly realized as a critical factor in the production of seedlings that continue to perform well, after they leave the greenhouse (Glerum, 1982). In contrast to germinant seedlings, and regardless of the photoperiod, older spruce seedlings do not free grow. For example, blue spruce seedlings three years old, or older, will not grow continuously under extended photoperiods while younger seedlings will (Young and Hanover, 1976). This change in shoot growth habit is important from standpoint of nursery management and plantation establishment. For example, understanding shoot growth phenology is important in controlling seedling frost hardiness which dramatically affects subsequent field survival and performancel(Glerum, 1982). As Romberger (1963) pointed out, although meristems are small, the whole course of a tree's development depends on the actions of these growth centers. In addition to being the ultimate source of the entire shoot, above the cotyledons, the shoot apical meristem is instrumental in the control of the growth and development of the subtending shoot (Kramer and Kozlowski, 1979). Thus, the shoot apical meristem is a potentially productive first place to look for ontogenetic and environmentally induced developmental changes. 4 The overall development of the spruce shoot and its responses to some environmental stimuli have been studied before (_e_.g_. Dormling gt a_l_., 1968; Pollard and Logan, 1977; Young and Hanover, 1976,1977a,1978). There appear to be no investigations of the initial developmental anatomy of the apical meristem in spruce, except the works of Burley (l966a,b), Gregory and Romberger (l972a,b), Cannell (1978a). None of these of these investigations involved comparison controlled environmental effects. This investigation attempted to quantify some of the environmentally affected some usmwwn mewapwwm mo uwnEsz u c .mxmmz cw 0mm mafiapmmm u fl I .mc0pmaauoo m>onm ucmwws EmumIcfimE @cfiacmmm n w "muonz \H th. mom. mva. mmvooo. + Ncmamo. I mmvmv. + vomm.o u and 5mm. Nam. mma. mammoo. I «Gnom. + Nmmm.a n and cum. mam. mew. amoea. + mmm~.~ u was mm mosumm smzuoz Ham. mam. oNH. mahooo. + ~.HH “Iwmm IIIIIIIIIIIIIIIIIIII omm. Nmm. ovm. mdomoo. I dmomm. + HwHH.o H wcq nmm. Hmm. mum. Gama. + amvm.a u you mm mosudm cabs»: E. .2. E. .238. - «mammwuam.-mmmmmdum .................... chm. mam. omH. Ndmmoo. I «mmmm. + MHH~.H u wca Nam. hum. Hon. «cowa. + mmmm.m u Mag mm mosumm msam mm \m \Mmmzm \flawcoe cofimmmumwm c mmfiowmm .mxmmz «m mom .OOwummouonm u: ¢~ Home: .wosumm >m3uoz 0cm .muflnz .Ufiunmn .wan mom Suzoum unmwmc mewavmwm onwummfioo now pound» mawpoe coMmmmummm .m.a wanna 24 As with the biomass data, a series of least squares polynomial regressions was developed to compare the height growth dynamics of the seedlings (Table 1;”. The original data was, again, log-transformed to homogenize sample variance among weeks. The result of these transformations is seen in Figure 1.38. In contrast to the biomass data, these height growth data represent the block means of repeated measurements of the same plants and the sampling error was, therefore, greatly reduced. The goodness of fit of regression for height increased considerably with the order of the fitted polynomial. There was a large and consistent decrease in the RMSE, which describes overall regression bias, with increasing polynomial order. This reduction in bias was greatest for hybrid and white spruces and was attributable to the greater change in curvature of their log-transformed data over time (Figure 1.38). The improvement in fit afforded by the third order equation was largely due to removal of the continuing downward trend due to the (negative) second order regression coefficient, at later measurement times. The seedling weekly mean relative height-growth rate (RHR) was calculated using weekly mean heights, illustrated in Figure 1.3A, by the same formula used to describe REE, substituting the natural logarithm of mean heights for those of biomass. These values are plotted in Figure 1.4. and represent the weekly-interval slopes of the log-transformed height data presented in Figure 1,38. 25 Figure 1.4 Weekly mean relative height growth rates (RHR) for spruce seedlings grown for 24 weeks under 24 hr photoperiod. A) blue spruce, B) hybrid spruce, C) white spruce, and D) Norway spruce. I I 4 2 3 2 2 2 D I I I I. r. MW 19‘ r. l R" l T. T “u n. n. a. I. R" 19‘ m” v. H" n. as a I I I II 7 I l I T! 6 I I I TI. I s I I II I I I I‘ I 3 I I I II 2 I I I I II I l I '0 I I I I I’ I I I I. 1| I I I7 I I I I6 I I! I Is r I I IO I I I I3 I I I I2 I BI CI II a a q q I q d - «quqqfiuuqqq - q 1 I 4 0 O 3 2 I o s 4 3 2 I o 1.9.765‘32Ios O 3 2 I O O C O I O I I C 0........ C O O O O o o o o o o o o 0 000000000 0 o o o o .xuux.tc\tc. uhcc tbxoco hzo_u: u>~b¢4u¢ 8cm: HEEKS 0F ORONTH 27 The slopes of the first derivatives of the second order polynomials show an experimentwise decline in 533. This decline is significant over the course of the experiment. This decline appears to be more asymptotic than linear, as implied by the better fit of the first derivative of the third order polynomial, which is a parabolic function. Linear regression models of needle number (PI) per unit height for each species were calculated (Table 1.4). PIs were derived for the unsampled seedlings, for each height measurement, using these models. Using these, interspecific growth rate differences appear smallem'(Figure 1.5A). Since these models are linear, the PI growth dynamics (Figure 1.53) hollow the same form as height (Figure 1.38). These results indicate interspecific differences in internode elongation existed over the course of the experiment. Table 1.4. Regression equations predicting plastochron index from height for each species. Species n Regression modell/ (r2) Blue spruce 46 Y = 18.58 + 1.84H .919 Hybrid spruce 51 Y = -5.64 + 3.22H .918 White spruce 51 Y = -10.57 + 3.08H .905 Norway spruce 46 Y = 26.61 + 1.96H .921 1/ Where: Y = Calculated plastochron index. H = Seedling height in mm above cotyledons. n = Number of seedlings measured. Figure 1.5 28 Seedling plastochron index (PI) for blue, hybrid, white, and Norway spruce seedlings grown for 24 weeks under 24 hr photoperiod. A) Mean weekly calculated (PI). B)!Nuanatural logarithm of weekly mean calculated plastochron index (PI). L00 BERN PLRSTOCHRON INDEX 29 — BLUE """ HYBRID "" HHITE "" NORHRY JA 1 l I l _aLUE . ----- HYBRID ”_ ---urm£ - ”'NORHRY '1 . gr" .37 57 .1 a TTlrlllllllllllllllllll 012 3 4 S 6 7 B 910H12131416161718192021222324 HEEKS 0F GROHTH 30 DISCUSSION The biomass and height data presented here, indicate that two different sets of dynamics are involved in their respective growth, over the same period of time. For biomass, over the course of the experiment, the Rafi was essentially constant. This indicates an exponential rate of biomass growth: W = W - ert Where: W = weight of the plant at time t. Wo= initial weight of the plant. t = the time of measurement. r = is the intrinsic growth rate. These types of equations imply that the growth rate of the organism increases in direct proportion with the mass. Thus, even small initial differences in mass have the same proportions at any stage. They tend, therefore, to provide a reasonable fit of the data only for small highly meristematic organisms. Since, however, no growth rate asymptote could be inferred from the data (Figure'l.2), use of biologically more "realistic", sigmoid functions, such as the Richards function (Causton and Venus, 1981), or splined polynomial regressions (Hunt, 1982) was precluded. The-REE values obtained in this study (Table 1;” averaged .32 week-1. This value is between those obtained by Grime and Hunt (1975) for Sitka spruce»(.22 week'l) and Norway spruce (.33 week‘l) germinant seedlings, for their initial five weeks of growth. Jarvis and Jarvis (1964) 31 reported different FER for germinant seedlings and 2—year-old seedlings of Norway spruce. For the germinant seedlings, the 'RER for the first 35 days after the cotyledon stage was .45 week‘1 in nursery grown seedlings, the rate was .03 week"1 during the period of shoot elongation. The initial dry weights of their two-year-old seedlings were less than those of 17th weekly sample weight of Norway spruce seedlings studied here, indicating that past growth history may have a major effect on'RER. Thus, the exponential growth phenomenon appears to be restricted to the continuous, early growth phase in spruce. Assuming that the RER was not different among species, and an apparent exponential growth over the course of the experiment, the most likely causes of the great differences in biomass are seed weight and germination time. Seed weights for blue and Norway spruce are generally higher than those of white and the hybrid. In spruce, seed size and embryo size are positively related (Burley, 1965). If an exponential growth rate is observed, these values become Wo in the equation above and, by definition, the Rafi is the same and constant, the biomass differences at any time are directly proportional to the seed weight differences. Long lasting seed weight effects are a fairly common observance (Sweet and Wareing, 1968; Evans, 1972; Hanover and Reicosky, 1972). Differences in germination rapidity also have a similar effect in the case of true exponential growth. By starting 32 their growth earlier, faster germinating seeds have a higher weight at whatever common time is chosen as to. In contrast to the biomass growth, seedling height did not follow an exponential pattern. From the second measurement on, seedling'RfiR declined. The asymptotic nature of the curve suggests that seedling height growth rate increases but, at a decreasing rate until some constant rate is achieved. A similar relationship between height growth and biomass growth was reported in Scots pine (Pinus sylvestris L.) grown from seed under 18 hr photoperiods (Oden and Dunberg, 1984). Main stem height is a function of the number and elongation of stem units (Doak, 1935), which are composed of needles and their corresponding internodes. Gregory and Romberger (l972a, 1972b) have shown that, for Norway spruce, the rate of needle formation and internode development rise towards a maximum during the first 160 days. They also note that internode development, at least in terms of vascular differentiation, seems to occur at a relatively constant chronometric rate (Gregory and Romberger, 1977). Under the cultural conditions used here, the average internode length for the hybrid and white spruce was significantly less than that of the blue and Norway seedlings. Other than the obvious possibility of genetic differences in this trait, one possibility is that the internal water potential varied among species, due to differing responses to the environmental conditions used. 33 Water potential deficits of less than 20%, induced by root pruning, have been associated with a 39% reduction in shoot elongation in 6-year-old white spruce (Marquard, 1983). If the interspecific root to shoot ratio differences seen in this study (Table 1.1) gave rise to a similar water stress effect, this might explain the shorter internodes. The shoot height differences seen may be largely due to interspecific differences in root growth potential in the environment used rather than inherent differences in internode growth potential. Within a species, differences in spruce shoot growth, in a common environment, seem more strongly related to stem unit number than internode elongation (Cannell, gt 31, 1976; Pollard and Logan, 1979). In conclusion, under optimum conditions, spruce seedlings of at least the three species and hybrid tested undergo an essentially exponential growth in biomass for at least 6 months. Although there seems to be no difference among the species in Rafi, there seems to be considerable interspecific plasticity, in seedling shoot morphology, and root to shoot biomass allocation. Height growth follows a different pattern with its increase tending towards a constant rate. This may be related to development of a constant rate of needle initiation and a finite potential for internode expansion. CHAPTER 2 THE DEVELOPMENT OF THE SPRUCE SHOOT APICAL MERISTEM IN A UNIFORM ENVIRONMENT ABSTRACT The development of the shoot apical meristem of blue, white x blue hybrid, white, and Norway spruce was studied. Seedlings were grown, in a controlled temperature room, for 24 weeks under a 24 hr photoperiod. Qualitative and quantitative measurements were made from median longitudinal sections by light microscopy. Quantitative measurements were made using a electronic digitizer and a volumetric computer program. The ontogenetic progression in apical development was similar for all species. Embryo apical meristems had fewer but larger cells that also differed qualitatively from those of the growing seedlings. In general, Norway spruce had the largest apices. There were no overall size differences among those of the other types. Apical meristems of all species increased in overall volume with age, apparently asymptotically. There were no significant species x age interactions. The relative volumes of the apical meristems in the apical initial-central mother cell zone, pith-rib meristem and peripheral zones changed during development. The relative 34 35 volume of the pith-rib meristem increased at the expense of those of the central mother cell and peripheral zones. Imbalances in the cytohistological zones of the meristem might lead to growth restrictions. 36 INTRODUCTION Blue spruce (Picea pungens Engelnn), Norway spruce (P; abies(L.) Karst.), and white spruce (_P_. glauca (Moench.) Voss) are spruce species that are of considerable commercial importance in the northern temperate zone. Seedlings of these species are often grown in greenhouses, using containers, frequently with artificially extended photoperiods. Use of these systems allows spruce germinant seedlings to be be grown to field planting size with a substantial savings of time over conventional nursery methods (9g. Hanover e_t _a__l_., 1976; Tinus and McDonald, 1979). To use these systems to their greatest advantage, it is necessary to understand how seedlings develop in controlled environments and to what degree different species differ in their responses. The photoperiodic protraction of indeterminate growth in spruce germinant seedlings is well documented (_e_._g_. Downs, 1962; Dormling st 31., 1968; Hanover and Reicosky, 1972). Less is known about the alterations in growth and development induced by these treatments. One of the most obvious alterations occurring under greenhouse culture is that of the ontogeny and phenolgy of the shoot apex. In nursery culture there are seasonal constraints on the development of the shoot apex. In order for the seedling to overwinter, shoot growth must stop and a bud must form as part of the overwintering process (Glerum, 1976,1982). The protracted indeterminate growth of spruce germinant 37 seedlings raised under long photoperiods allows more time for uninterrupted shoot formation and apical meristem development than either conventional nursery practices or natural field conditions. While the complete yearly phenology of mature spruce shoot apices, for some species, has been well described (Fraser, 1966; Owens and Molder, 1976; Owens 33 31., 1977; Pillia and Chacko, 1978; Thompsett, 1978; Harrison and Owens, 1983), the initial development of germinant seedling shoot apices, under natural conditions, has received less attention (Burley, 1966a). Much of the investigative work in this area has concerned bud development subsequent to seedling growth acceleration (Pollard and Logan, 1974b; Pollard, 1974a; Pollard and Logan, 1977; Young and Hanover, 1977a; Cannell and Cahalan, 1979). In spruce, there are fewer studies of the ontogenetic development of the apical meristem itself (Gregory and ~ Romberger, 1972a,b,1977; Cannell, 1978a). The apical meristem is traditionally defined as the undifferentiated tissue distal to the most recently formed foliar primordium. In their studies, the preceding authors developed mathematical models for apical meristem development and plastochron duration. Gregory and Romberger (1972a) also indicated that there was an ontogenetic progression in the development of the cytohistological zonation typical of conifers (53333 Foster, 1938). There have been many more investigations of germinant 38 seedling apical meristem development, especially with respect to cytohistological zonation, in the pines (Pings 33.) (Fosket and Niksche, 1966; Riding, 1972; Riding and Gifford, 1973; Mia and Durzan, 1974; Cecich, 1977; Kremer, 1984). In the pines, quantitative changes in zonation are seen as the seedling ages (Kremer, 1984) and in mature trees, during their annual growth cycle (Hanawa, 1966; Oweston, 1969). In mature pines there also appear to be differences within the crown of the tree in the size and internal structure of the dormant apical meristems (Tepper, 1963). As spruce seedlings develop under the lengthened photoperiods used in most greenhouse accelerated-growth seedling production systems, they undergo size rlated changes in growth rate and apical activity (Chapter 1). This experiment investigated the quantitative changes in apical meristem size and histological development of three species and one interspecific hybrid of spruce to test for relationships between apical meristem development and seedling shoot development. MATERIALS AND METHODS Seed source and cultural system Blue, white, white x blue hybrid, and Norway spruce seedlings were used in this experiment. The sources were the same as those described in Chapter 1. As before, the hybrid will hereafter be considered as a fourth species, for 39 convenience. Seeds were sown in plastic cases of 5 x 5 x 27 cm polyethylene coated paper plant bands, filled with a 1:1:1 sphagnum peat: perlite: vermiculite mixture. These were distributed randomly within the blocks of a larger growth study (Chapter 1). Nutrient application and maintenance were as described in Chapter 1. Gross morphology At weekly intervals, starting 5 weeks after sowing, 12 seedlings (one of each species in each block) were randomly selected for morphological and anatomical analysis. After excision of the apical region for anatomical analysis, the root systems were gently washed free of potting medium and cut from the shoot. Roots and shoots were both oven dried at 60°C then weighed to the nearest mg , for total biomass and root to shoot ratio determination (Chapter 1). Main stem needle and primordia number (Plastochron Index (PI)) were also recorded. Microtechnique The shoot apical meristem and upper 3 to 5 mm of stem of each of the seedlings measured above were excised and immersed in FAA (formalin:acetic acid:alcohol:water), to kill and fix the tissues. This was done under partial vacuum to remove trapped air. They were then dehydrated in a water:ethanol:t—butanol series (Johanson, 1940) followed by infiltration with Paraplast embedding medium. Serial longitudinal sections were made at 10 pm and those passing 40 through the apical meristem were collected and mounted on glass slides. The dewaxed slides were progressively stained with hemalum (Sass, 1958) and regressively with safranin (Johanson, 1940). In addition to the sampled seedlings, three embryos of each of the species used were also excised from dry seeds seeds and prepared as above. Median longitudinal sections were identified for measurement. Photomicrographs were taken with a Leitz Ortholux microscope using a 10X plano objective and a 10X wide field occular with a Wilde 35mm camera system. Constant magnification enlargements, at 330 X, were made from these negatives for digital analysis. Selected material was also photographed with the same microscope system usng a 4 x 5 inch plate camera. In all cases, calibration photomicrographs were made using a micrometer slide. Volumetricanalysis Digitizing of the apical meristem photographs was done with a GTCO micro-datizer digitizer coupled to a CDC Cyber 750 computer, by "tracing" the outlines of the Whole meristem and pictured cytohystological zones with the digitizing cursor. This initial process yielded two dimensional coordinate data with a Y value recorded for each 10 scaled micrometers (3.3 mm) in the X direction (Figure 2.1). Total meristem volume and cytohistological zone volumes were calculated from these data with a FORTRAN program, on the same computer. Figure 2.1 41 Median longitudinal section through the shoot apical meristem of a representative 16-week-old Norway spruce seedling. A) Photomicrograph (330X). The meristem base, B) as determined by the level of the last formed primordium, and the cytohistological zones have been highlighted to show their boundaries. A digital rendition of the same meristem generated by plotting the raw data from tracing the highlighted areas in (A). The digitizer was set to record the ordinate "Y" value on every change of 10 scaled pm in the abscissa "X" direction during tracing. The procedure was repeated for the boundary of each zone, cmz (central mother cell zone), rpm (pith-rib meristem), and the pz (peripheral zone). \m‘.‘ o f’ box 0': / \f-u" \ DZ / *4 pz / rpm CI O l , “\ n \ 0 II"! I I‘ll -200 -150 -100 -50 0 50 100 150 200 mzmqmit omranm z~ maaz~oaaan < .— c o I X COORDINRTE IN SCRLEO fl HETERS 43 Volumes of the whole meristem and those of the individual cytohystological zones, were calculated by summing mean conic frustra calculated from the digitized data. In the case of the total meristem volume, for example, the program calculates the volumes of two series of frustra. The first series progresses acropetally, starting on the left side (negative most X value) at the level of the last formed needle primordium and ending with a broad cone at the top of the meristem. The second is a basipetal series starting at the top center of the meristem and progressing downward to the same base line. The incremental volumes of each series are halved and summed to accommodate asymmetrical apices and variation in Y axis positioning. The algorithm uses the following equation to calculate incremental half frustra: Xmax V= E l/2(l/3th(Ri+R§+RlxR2) i=Xmin Where: Xmin = The X coordinate of the left most point of intersection of the curve of the apical meristem surface and the base line described above. Xmax = The x coordinate of the right most point of intersection. h = The height of the frustrum. 44 R1 = The radius of the bottom of the frustrum (The first value is thus Xmin). R2 = The radius of the top of the frustrum (The first value is thus Xmin + 10). The volumes of the cytohistological zones were calculated by decomposition into sums of similar series. The apical initials and central mother cell zones were considered as one zone (central mother cell zoned, based on their position and morphological similarity (Owens gt al., 1977; Mauseth and Niklas, 1979). Measurements of mean basal radius and apical height were also recorded. RESULTS Initial observations The apical meristems of the dry embryos were strikingly different from those of the first sampling of germinant seedlings. The cells of all embryo apices were larger (based on cross sectioned area) than those of the first sampled germinant seedlings. The cytoplasm was filled with numerous and relatively large U.tx>6 pm) weakly staining bodies (Figure 2.2A), which did not stain darkly with iodine, as would starch. The nuclei, while staining deeply with safranin, were smaller and had a distorted appearance. The cell walls also appeared wrinkled and stained more densely Figure 2.2 45 Median longitudinal sections through the apical meristems a of a spruce embryo and 5-week—old seedling. A) Representative spruce embryo (blue spruce). B) Note large, irregular cells with densely staining cellwalls, frequently distorted nuclei, and largeweakly staining cytoplasmic bodies (seearrow). Bar = 50 pm. Representative S-week-old seedling (hybrid spruceL.Note smaller cell size, more regular nuclei, loss of cytoplasmic bodies, mitotic activity (seearrow) and smoother, lighter staining cell walls. Bar = 50 pm. 47 than those after germination. There were few qualitative differences observed among the cells of the embryo meristems. The cells in upper center of the meristem lacked the vacuolization seen in the apical initials and central mother cells of mature apices. Those cells along the flanks of the meristem were slightly smallem'and less elongate than those along the vertical axis. The alignment of the cell walls was, in general, suggestive of pattern of zonation typically seen in apical meristems of actively growing conifers. Growth and development By the first sample, 5 weeks after sowing, meristems of all species were composed of more, but smaller, mitotically active cells. There were pronounced files of pith-rib meristem cells and in some cases, a distinguishable central mother cell zone, with lighter staining nuclei and a vacuolated appearance was evident. The large, weakly staining bodies seen in the embryo meristems were completely absent (Figure 2.2B). The nuclei and cell walls, as well, were more normal in appearance. Cytohistological zonation became increasingly distinct for all species through the first 8 to 12 weeks after which there appeared to be little change (Figures 2.3-2.6). There was one significant interspecific difference in total apical meristem volume. Norway spruce apical meristems were significantly larger then the other species, when averaged over the course of the experiment (p<.05%. Figure 2.3 Median longitudinal sections through the shoot apical meristems of blue spruce seedlings at different times during their development: A) From an embryo, excised from a seed. B) 5 weeks after sowing. (D 6 weeks after sowing.[n 8 weeks after sowing. E) 12 weeks after sowing. F) 16 weeks after sowing. G) 20 weeks after sowing. H) 24 weeks after sowing. Bar length = 100 pm. Sections were cut at 10 pm. Figure 2.4 50 Median longitudinal sections through the shoot apical meristems of hybrid spruce seedlings at different times during their development: A) From an embryo, excised from a seed. B) 5 weeks after sowing. (D 6 weeks after sowing.[n 8 weeks after sowing. E) 12 weeks after sowing. F) 16 weeks after sowing. G) 20 weeks after sowing. H) 24 weeks after sowing. Bar length = 100 pm. Sections were cut at 10 pm. Figure 2.5 52 Median longitudinal sections through the shoot apical meristems of white spruce seedlings at different times during their development: A) From an embryo, excised from a seed. B) 5 weeks after sowing. (D 6 weeks after sowing.ID 8 weeks after sowing. E) 12 weeks after sowing. F) 16 weeks after sowing. G) 20 weeks after sowing. H) 24 weeks after sowing. Bar length = 100 pm. Sections were cut at 10 pm. Figure 2.6 54 Median longitudinal sections through the shoot apical meristems of Norway spruce seedlings at different times during their development: A) From an embryo, excised from a seed. B) 5 weeks after sowing. (D 6 weeks after sowing.tn 8 weeks after sowing. E) 12 weeks after sowing. F) 16 weeks after sowing. G) 20 weeks after sowing. H) 24 weeks after sowing. Bar length = 100 um. Sections were cut at 10 pm. 56 There was no detectable overall difference among the blue, white, or hybrid apical meristem volume. In addition, there was no significant age x species interaction, indicating that the course of apical development was similar for all the species under the environmental conditions used. The volumetric data was, therefore, pooled over species for further analysis. Apical meristem volume increased most, early in the experiment. ‘With the exception of the mean value for week 21, there were no significant differences in volume after week 10. Total meristem growth, thus, occurred in an apparently asymptotic manner (Figure 2.7A). Meristem volume was more highly correlated with seedling plastochron index (PI) than chronological age (Table 2.1). This is also supported by the more stable maximum volume, usually 5 to 6 million pm3, Shown in Figure ZJHL. The overall shape of the apical meristem also changed over time, increasing more radially than in height through the first 10 weeks or PI 200 (Figure 2.7C-D). The meristem thus became broader during early development (Figures 2.3-2.6) and radius was a better linear indicator of apical development (Table 2.1) than height. Within the meristems themselves, the proportion of tissue in each cytohistological zone changed continuously. The biggest change was seen between the peripheral zone and the pith-rib meristem relative volumes (Figure 2.8A-D). The relative volume of the pith-rib meristem increased and that Figure 2.7 57 Spruce seedling shoot apical meristem volume, radius and height development under 24hr photoperiod. A) Total apical meristem volume averaged over blue, hybrid, white, and Norway spruce species for the first 24 weeks. B) Total apical meristem volume averaged over blue, hybrid, white, and Norway spruce species at intervals of 50 plastochrons. C) Apical meristem basal radius and height averaged over blue, hybrid, white, and Norway spruce species for the first 24 weeks. D) Apical meristem basal radius and height averaged over blue, hybrid, white, and Norway spruce species at intervals of 50 plastochrons. 58 7000 - 1 1 q q H . 1 u 4 . D “.00--.. I I . I c o I C.--“ I a I o .0...- I c o I .00. l u I q... r o I 8... . ....... L I UM . I I .P D! u 1 RE . I R" q ........ ‘ I o o I c o r _ m _ .. .1. I w _ _ 1 f I 0......DOEO- c I \V hunch. I 0| IIIIII I I I I I I I f. .l 8 I U I m I I a I R I . _ . I 00 d u d u T d d d d o o o o o o 0 0 0 0 0 0 W w 0 W w 0 0 0 0 0 0 1 1. M l B s ‘ a 2 I. a .no ans: . 2.33 55:3: .52.... a z: . 5.5—u: 9:: was: 4330 tuhuucuz accuse 100 200 300 400 800 000 10 12 14 10 10 2022 24 0 NEEKS 0F ORONTH PLNSTOCHRON INDEX Figure 2.8 59 Relative volumes(Mfcytohistological zones within the developing shoot apical meristems of blue, hybrid, white and Norway spruce grown for 24 weeks under 24 hr photoperiod. A and B) Relative rib-pith meristem volume related to age and PI respectively. C and D ) Relative peripheral zone volume related to age and PI respectively. B and F ) Relative central mother cell zone volume related to age and PI respectively. 60 I I I I 1 I TI Ifi I 0 wtmtmmmmmmmmmm PLMIW non » curacy. 3808:; U323: d ‘ . 1 n 1 s Q! .N. gig in gags - o 7 d ‘ I. .N. 958» 2» 4.50 :3»: 5.33 II- 202224 I I to I. I I4 KERI N WIN 61 of the peripheral zone decreased significantly. Table 2.1 Spearman rank correlations between shoot apical meristem developmental characteristics and selected growth indices for pooled spruce apical meristem data. Meristem Meristem Growth Characteristic PI Age Volume Rate l/ Total Volume .62 *F? .54 *** I; - - .53 *** PRM / Totalz/ .6l *** .64 *** .48 *** .47 *** PZ / Totali/ -.46 *** -.45 *** -.l6 ns. -.42 *** CMZ / Totali/ -.09 ns. -.11 ns. -.32 *** -.01 ns. Basal Radius .58 *** .50 *** .94 *** .50 *** Height .43 *** .38 *** .80 *** .39 *** :;*>— Eggnificant at the P = .001 level; ns. not significant l/ Previous week's mean height growth rate. 2/ Relative pith-rib meristem volume. 3/ Relative peripheral zone volume. 4/ Relative central mother cell zone volume. There was no significant decrease in the central mother cell zone relative volume, over the course of the experiment. At higher PI however, there may be a loss, as indicated in Figure 2.8F. Based on the volumetric approach used here, apical meristem development is more strongly related to PI than chronological age, under the continuous growth conditions used. In addition, while correlations between the volumetric attributes measured here and height growth rate (in the week prior to harvest) are similar to those for PI and age, they are weaker. When the relative cytohistological zone volumes are compared with the total 62 apical meristem volume, a reversal of the significances in relative volume loss between the central mother cell zone and the peripheral zone is seen. In other words, central mother cell zone relative volume loss is more strongly associated with increasing total meristem volume than it is to time or PI (Table 2.1). DISCUSSION The changes seen between the dry embryo apices and those of the first sample (at 5 weeks) are similar to those previously reported during germination in Norway spruce and other conifers (Tepper, 1964; Foskett and Miksche, 1966; Gregory and Romberger, 1972a; Riding, 1972). The large, weakly staining bodies prevalent in the embryonic apices showed no birefringence under polarized light and did not stain positively for starch, with iodine. Although they did not stain very darkly with hemalum (hematoxylin) they otherwise fitted the description of the inclusions seen before in spruce embryos. These appear to be protein-like in nature; probably storage proteins (Gregory and Romberger, 1972a). Similar types of bodies were reported in the embryos of Monterey pine (Pinus radiata Laws.) by Riding and Gifford (1973) and in ponderosa pine (g. ponderosa) by Tepper (1964). Mia and Durzan (1972) investigated the ultrastructural changes in germinating jack pine (_P_.banksiana) embryos and noted that there was a progressive loss of protein-like bodies in the meristematic 63 cells during germination. These bodies are 50% digested within 48 hr of imbibition (Cecich and Horner, 1977). The condition of the embryonic nuclei, suggestive of an inactive state, appears to have been similar to that seen by Riding and Gifford (1973) in Pinus radiata. The distortion seen in their study and this one were less apparent in several other studies on germinating conifers gyg, Gregory and Romberger, 1972a; Mia and Durzan, 1974). This may have been due to the different fixatives used or seed storage conditions. The FAA used here and in Riding and Gifford's study may have caused less swelling during fixation due to the ethanol content. Some swelling, as indicted by elongation of the excised embryos, was present even with the fixative used here. In contrast, the cells of growing meristems show some shrinkage due to FAA fixation (3.3. Figurer2.ZB). Since all meristems were treated alike, however, these differences should not affect comparative differences (Gregory and Romberger, 1972a). The great reduction in cel 1 size seen by the first sample time appears to be largely due to the exhaustion of the protein bodies and the resumption of mitotic activity associated with needle initiation. In general, the first few days of post imbibition development seem to be more involved with "reactivation" of the shoot meristem rather than active primordia initiation. The occurrence of an apparently asymptotic form of growth of the apical meristem has been seen previously in 64 spruce. In Gregory and Romberger's study (l972a), the basal radius of the meristem increased, but at a slowing rate, for 168 days under continuous growth conditions. Cannell (1978a) reported similar growth dynamics in the apical meristems of Sitka spruce (Picea sitchensis) grown under long days. The results presented here are in closer agreement with those of Gregory and Romberger. Their data indicate that a relatively stable basal diameter of about 300 pm is reached within 100 days. While Cannell (1978a) reported somewhat larger sizes for Sitka spruce, he suggests that they may have been due to higher light intensities. As seen in Table 2.1, basal radius appears to be the best linear estimator of apical volume. In their work with Monterey pine Riding and Gifford (1973) reported that there was no observed difference in apical meristem histochemistry from 84 days until the end of their experiment at 12 months, using greenhouse culture. Previously, Riding (1972) reported that cytohistological zonation as established by 60 days after sowing remained the same for at least the next 18 months. Total volume, after an initial decline during the first 10 days increased during the rest of the experiment. Kremer (1984) found, however, using measuring techniques similar to those employed in this study, that the volume of the apical meristems of jack pine (Pinus banksiana) increased up to about 60 days after sowing then decreased in volume for the remaining 28 days. He mentions that this was perhaps due to a growth chamber 65 misfunction. In his experiment, the reduction in total meristem volume was associated with an increase in plastochron duration (lower stem growth rate). In the first 24 weeks of growth, spruce seedlings, grown under optimal conditions, appear to initiate needles in an asymptotically increasing manner (Chapter 1). This implies a corresponding asymptotic decrease in plastochron duration occurs (Gregory and Romberger, 1972a,b, Cannell, 1978a; Cannell and Cahalan, 1979). Shoot growth, based on the formation and development of stem units at the apical meristem, is thus controlled partly by the size of the meristem. The formation of a primordium at the meristem results in the loss of tissue from the peripheral zone which must be replaced, by mitotic activity, in order to perpetuate the meristem. Thus, the meristem limited rate of shoot growth is also determined at least in part by the tissue volume partitioned into the primordia (Romberger and Gregory, 1977; Cannell, 1978a), the peripheral zone volume and peripheral zone cell cycle time. An interesting developmental trend that has not been addressed in previous studies of spruce germinant seedlings: is that of the continuing change in apical meristem zonal distribution seen in this study. These were similar to those seen in jack pine (Kremer, 1984). In that study, apical meristem volumes were calculated using parabolic periods of revolution, an alternative approach to the "disk 66 method" used here. Kremer found that the peripheral zone relative volume decreased and the pith-rib meristems relative volume increased for the first 68 days. While PI was the growth index most closely associated apical meristem volume»(Cannell, 1978a; Gregory and Romberger, 1972a), its relationship with the cytohistological zone trends seen in this study is less clear. Changes in the central mother cell zone, seem more strongly related to overall meristem volume than PI although the trends are the same. To a lesser extent, rib-pith meristem relative volume may be more strongly related to age. The relative volumes of the cytohistological zones in the shoot apical meristem may be involved in hormonal output from the shoot apical meristem (Mauseth, 1979). This may also affect the determination of primordium fate and other aspects of the seedlings physiology. In addition, if the rate of peripheral zone loss were to continue according to the trend reported here, it might present a physical limit to primordium initiation and thus to continued free growth. CHAPTER 3 THE EFFECT OF SHORTENED GROWTH CYCLE NUMBER ON SPRUCE SEEDLING APICAL DEVELOPMENT AND FREE GROWTH POTENTIAL ABSTRACT Seedlings of blue, white x blue, white and Norway spruce were grown in a shortened growth—cycle (SGC) regime which exposed them to five, consecutive, artificially shortened, cycles of shoot growth and bud formation. Seedlings of all species were able to resume free growth in a propitious environment, even after four such cycles. Seedling development under SGCs was much reduced compared to normal nursery-grown seedlings or seedlings grown under accelerated conditions for less than one year. The average seedling height at the end of five SGC was less than that of an average production-run two-year-old (2+0) seedling. The main stem buds, sampled at the end of each growth cycle, were smaller than normal seedling buds. The stage of phenological development at the end of each cycle seemed to decline with cycle number. While meristem volume increased: there were progressively fewer needle primordia in the buds. This may indicate that as seedlings get bigger more time is needed for complete bud formation. 67 68 I NTRODUCT ION Mature spruce (Picea sp. UL) Dietr.) trees undergo an annual growth cycle in which the annual shoot growth results from the elongation and expansion of preformed stem units existing in the overwintering bud. A stem unit consists, u v w4o>u _ _ m wgoru _ N wqu>u d m...u>u quo>u ow wt... oulo ww._u>o om ~50."— sunk ww40>o ow mwmz... 02.4 wm4u>u am 0:... Tim u40>u om uzo I mm om m5 on: mN~ om— m: cow mNN ( HN ) lHOIBH .lOOHS ONI'IOBBS 80 weeks. Buds were seen on all SGC seedlings by the fourth week of the cycle. Thus, all seedlings had terminal buds formed, prior to the start of the chilling period of the cycles, and height growth had ceased. Apical.development No significant interspecific differences in the total apical meristem volume were observed among SGC seedlings. There were, however differences in total apical meristem volume among the first four sequential cycles uraOS). The buds from the fifth cycle were accidentally dried and were thus useless for apical meristem volumetric measurement. There was an increase in total meristem volume with cycle number (Figure 3.3, Table 3.1%. No significant differences were seen, either among cycles or species, in the relative volumes or the cytohistological zones. Cytohistological zonation was indistinct within the dormant meristems. The apical meristems of SGC seedlings, even after four cycles, were significantly smaller than those of the two- year-old and older nursery grown and mature blue spruce apical meristems (Table 3.1). The SGC meristems were not significantly larger than the one-year-old nursery grown seedling apical meristems even after four growth cycles (3.1. Figures 3.4 and 3.5). Figure 3.3 81 Representative median longitudinal sections through the terminal shoot bud of blue spruce seedlings after 1 to 4 shortened growth cycles. A) one cycle, B) two cycles, C) three cycles, and D) four cycles. Bar = 300 pm. Thickness= 10 pm. Figure 3.4 83 Representative median longitudinal sections through buds of spruce seedlings after four shortened growth cycles. A) Blue spruce, B) Hybrid spruce, C) White spruce and D) Norway spruce. Bar == 300 pm. Thickness = 10 pm. 85 Figure 3.5 Representative median longitudinal sections through naturally-cycled, dormant blue spruce buds. A) 1+0 nursery grown. B) 2+0 nursery grown. C) 2+1 transplant. D) Mature tree (upper crown). Bar = 500 pm. Thickness = 10 um. 87 Table 3.1 Apical meristem volumes and needle primordia seen in spruce buds developed under SGC and in nursery-grown and mature buds. All Species Pooled Blue Spruce (shortened cycles) (nursery grown) Cycle Meristeml/ Needlesg/ Seedling Meristem Needles Number volume in bud age volume in bud l ll48a3/ 13.2ab 1+0 2658a 14.7_ 2 2258ab 15.5b 2+0 6019b 24.9 3 1574ab 12.6a 2+1 4140 26.0 4 2597b 9.0a 50+ 8745 28.0 5 —--- 8.4a --- —--- ---- 1/ Meristem volumes expressed in pm3x106. 2/ Needles seen in median longitudinal section. 2/ Means within a column followed by the same letter are not significantly different at the (p =.05) level, Duncan's mu tiple range test. With the exception of the first cycle, the number of needle primordia within the buds, seen in cross section, declined with successive cycle number (Table 3.1, Figure 2L3). In buds from all cycles, Norway spruce had significantly more needles than any of the other species. There were no other interspecific differences. ‘All species responded similarly to the sequential cycles. The number of needle primordia within the buds of the SGC blue spruce was never significantly more than that seen in one-year-old, blue spruce, nursery grown seedlings (Table 3.1, Figure 3.3). In addition, the number of needle primordia in two- 88 year-old and older nursery grown blue spruce was greater than that of the one-year-old seedlings (p<.01) or any SGC seedling. DISCUSSION The average shoot growth under the long-day greenhouse conditions (free growth test) appeared to be asymptotic in nature (Figure 3JD. Under optimal conditions, spruce shoot height-growth often increases in a nearly linear manner (31. Dormling 31 31., 1968; Young and Hanover, 1977a; Cannell and Cahalan, 1979; Chapter 1). The decline in growth rate seen in the free-growth test material here (Figure 3.2) may have been induced by the high temperatures and/or water stress encountered in the green houses during the summer. Temperatures in mid and late summer were often in excess of 30°C. These have generally been found to be too high for optimal spruce growth (3.3. Young and Hanover, 1978). This combined, with a higher light intensity, made maintenance of uniform water status difficult. Based on the shortened growth cycle system used in this study, the developmental factors which control the free growth propensity in spruce seedlings do not seem to be bound to the number of growth cycles, p35 33. There were, however, a number of quantitative differences between SGC seedlings and those cyclically grown under natural 89 conditions, which need to be considered when interpreting the SGC effects. First, although the seedlings in this experiment were subjected to up to 5 phenologically complete growth cycles, the mean height of the seedlings averaged only 88 mm at the end of the fifth cycle UH». This is considerably smaller than the height of an average S-year-old nursery-grown spruce seedling of any of the species used. iFor blue spruce, even the tallest of the fifth cycle seedlings (132 mm) was only about average for two-year-old (2+0) commercial nursery stock (3:. Chapter 4). In most past studies of free growth inducibility in various ages of spruce, seedling morphological or developmental characteristics, other than age, have not been reported (Nienstaedt, 1966; Young and Hanover, 1976; 1977b)- It was illustrated by Pollard (1974b) that seedlings, in a free growing state, became increasingly sensitive to a dormancy inducing stimuli (an intervening two weeks of 8 hr photoperiods) as they aged. However, the relationship between height and dormancy (no resumption of shoot growth) appeared to be stronger (r2=.90) than that of age and dormancy (r2=,72), The results reported here indicate that the consideration of seedling height, in age-class seedling development tests, may warrant further attention. A second major difference seen between the development of the SGC seedlings and those of nursery-grown and mature blue spruce, is that of terminal bud development. In 9O naturally grown spruce buds, seedlings or trees older than one year generally have far more preformed needles in the buds (Table 3.1, Figure 3.5) than do one-year-old seedlings (Young and Hanover, 1977b; also 31. Burley, 1966a and Owens and Molder, 1976). With the SGC seedlings, as indicated in Table 3.1 and Figures 3434L5, there was an overall trend among all species towards fewer needle primordia with each successive cycle. This suggests that with progressive cycles, either more time was necessary for bud scale formation prior to needle primordia initiation or the needles were initiated at a lower rate. From the number of scales seen in longtudinal sections, it appeared that more primordia developed as bud scales in early part of later cycles (31. Figures 3.3A and 3.3D), supporting the former of these two possibilities. The dormant apical meristems in the SGC material were generally smaller than that of typical one-year-old nursery- grown blue spruce (Table 3;”. They were also smaller than those reported in one-year-old nursery-grown Sitka spruce (Burley, 1966a) or four-year-old dormant white spruce (Cecich and Miksche, 1970). They never attained the size of any of the older naturally grown blue spruce meristems. The SGC meristems were also smaller than meristems from six— month-old spruce, from the same seedlots, growing under free growth conditions (Gregory and Romberger, 1972a; Cannell and Cahalan, 1979; Chapter 1). 91 It is important to note, with regard to the last observation, above, that there are fluctuations in meristem size during the natural growth cycle. In the annual cycles of the spruce species that have been observed, the apical meristem reaches its maximum size between the end of bud scale initiation and late needle-primordia initiation. This is followed by a decline until late autumn, when the meristem is at its smallest size until the first bud scales are initiated prior to budbreak the next year (Cecich and Miksche, 1970; Owens and Molder, 1976; Owens 33 31., 1977; Pillia and Chacko, 1978; Thompsett, 1978). A similar increase and decrease in meristem size was seen, during photoperiodically induced budset, in Sitka spruce germinant seedlings (Cannell and Cahalan, 1979). Thus, it is important to consider the phenological stage of the material observed, when making quantitative comparisons of apical meristem volume. In this experiment, in light of the decreasing number of needle primordia seen within the buds, the size increase of the apical meristem seen may or may not reflect the actual ontogenetic developmental progression. If primordia formation were interrupted by the beginning of the chilling treatment used in this experiment, it might have left the apical meristem larger than if needle initiation had proceeded to completion. Finally, the bud burst phenology of the SGC seedlings also differed from that seen in spruce seedlings having the 92 equivalent number of natural Cycles. Through out the experiment, budburst had occurred on all seedlings prior to the return to 8 hr days in each cycle. In one—year-old (1+0) through seven-year-old (2+2+3), completely chilled, white spruce seedlings brought under 18 hr photoperiods, budburst ranged from 26 to 39 days (Nienstaedt, 1966). Young and Hanover (1977b) found a similar age-related increase in time to budbreak in blue spruce seedlings. However, when seedlings of white, blue, and Norway spruce were grown under long photoperiods (20 hr) for 4 months, allowed to set bud for 2 months under 13 hr photoperiod then chilled for 6 weeks under 13 hr photoperiods the buds broke 14, 16, and 25 days later, respectively, when the seedlings were returned to favorable growth conditions (Nienstaedt, 1967). Similar results were seen with blue spruce when the initial growth time was varied from 2 to 6 months (Young and Hanover, 1977). The results of this study are in general agreement with these latter observations, indicating that perhaps the artificial shortening of the bud development period effects the depth of the subsequent dormancy as well as shoot growth potential. In conclusion, while spruce seedlings can be "forced" through almost 4 artificially shortened growth cycles in the time filled by one natural cycle, there are quantitative differences in the phenological changes usually associated 93 with these cycles. Growth-cycles accumulated, 33333 stricto, do not appear to be developmental characteristics intrinsically involved with loss of free growth in spruce. Since size alone does not appear to be a limiting aspect (Dormling 33 31., 1968; Chapter 1), one possibility is that the developmental stage past which free growth will not occur in spruce involves some interaction of age and size. CHAPTER 4 EFFECTS OF SEEDLING SIZE, AGE AND CULTURAL METHOD ON SUBSEQUENT PHOTOPERIODICALLY-MEDIATED GROWTH IN BLUE SPRUCE SEEDLINGS ABSTRACT Blue spruce seedlings of three size classes, and two cultural methods, "accelerated growth" (AG) and "conventional nursery" (NUR), were growth-tested under 24 hr long day (LD) and 12 hr short day (SD) photoperiods for 16 weeks, in a temperature controlled room. AG seedlings initially had lower overall weight, lower plastochron index, smaller stem caliper, and fewer preformed needles in their buds than did NUR seedlings, within size classes. AG seedlings broke bud sooner than the NUR seedlings, perhaps due to their age or the storage conditions. Seedling growth phenology was evaluated in two phases, flush growth and subsequent growth. Photoperiod and seedling initial height had no significant effect on the time to bud-break or shoot flush rate. NUR seedlings grew faster during the flush phase and had greater total flush height. The internode elongation was less than that of the AG seedlings, however. After the flush phase AG seedlings of all sizes grew faster than NUR seedlings, under LDs. The smaller two size classes of NUR seedlings grew faster than 94 95 the largest, which showed no significant post flush height growth. Fewer free growth needles were formed on NUR than AG seedlings, all of which free grew under LDs. Some NUR seedlings did not resume free growth. Lack of free growth, as determined by needle counts, did not appear to be related to initial height. It appeared that a lower growth potential, largely due to root disturbance and low photosynthetic photon density, more strongly affected free growth induction than did initial seedling size or age. 96 INTRODUCTION Along with increased size, aging spruce seedlings show changes in shoot development and phenology. Under nursery conditions, spruce germinant seedlings grow indeterminately in their first year until environmental stimuli, largely photoperiod and temperature, induce the formation of an over-wintering bud (3.3. Burley, 1966b; Arnott, 1974; Powell, 1982). In following years, spruce seedling shoot growth changes from this completely indeterminate pattern of growth ("free growth" (Jablanski, 1971)), to a completely determinate type of shoot growth (Jablanski, 1971; Pollard and Logan, 1976; Young and Hanover, 1976; Powell, 1982). In this mature growth pattern, all of a years needles and their internodes exist, in a "telesc0ped" manner, within the over- wintering bud formed in the previous year (Fraser, 1966; Owens and Molder, 1976; Owens 33 31., 1977; Bongarten, 1978; Pillia and Chacko, 1978; Harrison and Owens, 1983). This change of growth pattern, in the species in which it has been observed, is usually completed within two to five years (Nienstaedt, 1966; Jablanski, 1971; Young and Hanover, 1976; Powell, 1982). In blue spruce (31333 pungens Engelnu), for example, seedlings in the nursery appear to have a completely determinate growth pattern by their third growing season. This seems to be the case for most spruce species, although some sources of black spruce (Picea mariana (Mill.) B.S.P.) may retain the free growth 97 characteristic for up to 12 years (Pollard and Logan, 1976). In addition to the loss of free growth under natural conditions, there seems to be a concomitant change in their response to extended photoperiods. For example, germinant seedlings and one-year-old blue spruce seedlings resume free growth when placed under 24 hr photoperiods. Three-year-old and older seedlings set bud at the end of shoot elongation (Young and Hanover, 1976). A similar lack of free growth response was reported in five-year-old white spruce and thirty-year-old grafts (Neinstaedt, 1966). Under extended photoperiods, two-year-old, nursery- grown blue spruce seedlings are intermediate in their shoot growth response. They exhibit three patterns of shoot growth. After flushing and elongation of the preformed shoot, these seedlings either continue needle formation and stem elongation (free growth), set bud then flush and resume free growth, or set bud and remain dormant (Young and Hanover, 1976). Similar shoot growth patterns were seen in white spruce (Watt and McGregor, 1963; Nienstaedt, 1966) and Norway spruce (Farrar, 1961) of a similar age. These responses imply that such seedlings are in a transitional stage in the development of their photoperiodic response. In order to determine which, if any, of the observable developmental changes seen in early seedling ontogeny are involved in the changes in photoperiodic response, it is necessary to separate and test them. Accumulation of growth cycles, alone, does not seem to be the limiting factor 98 (Chapter 3). Size, alone, also does not seem to limit free growth, since accelerated seedlings can be grown to larger sizes than typical non-responsive nursery seedling (3.3. Dormling 33 31., 1968; Hanover and Reicosky, 1972; Heide, 1974a; Chapter 1). Since two-year-old nursery—grown seedlings have shown an intermediate response, they are an appropriate population to analyze for developmental attributes associated with free growth propensity. In addition, since one-year-old, similar-sized, accelerated-growth seedlings arelavailable, some insight can be gained into interactions of shoot size and age by comparing them to similar sized nursery-grown seedlings. This study was designed to test the relationships between size and age on photoperiodically-induced shoot free growth and seedling development in blue spruce. MATERIALS AND METHODS Blue spruce seedlings, probably from southern Colorado seed sources, were obtained from a commercial nursery in lower Michigan. Two-year-old (2+0) conventional nursery stock (NUR), fall-lifted and stored bare-rooted, was used along with one-year-old, accelerated-growth, container-grown plug seedlings (AG). The seedlings had been in protected cold-storage, since lifting in the fall. All seedlings were sorted into three size classes, 75-150 mm, 150-225 mm, and 99 225-300 mm, based on height from the cotyledons to the tip of the terminal bud. Individual seedling heights, to the nearest 5 mm, were also recorded. The seedlings were received on March 31 and stored at about 4°C until April 4 when all seedlings were transplanted into 10 x 10 x 27 cm, polyethylene-coated paper plant-bands filled with a peat:perlite:vermiculite (1:1:1 by volume) mixture. Each band then received an equal amount of fertilizer (Peter's 19:18:17 (NPK) and STEM trace minerals) solution, and the whole experiment was watered to drainage. Additional fertilizer and water were periodically applied during the experiment, uniformly and simultaneously to all seedlings, as needed. Two photoperiods, short day (SD) 12 hr and long day (LD) 24 hr were used in this experiment. Irradiation was provided by 96 inch VHO fluorescent lights. Two lamp fixtures (with two tubes each) were used in the (SD) photoperiod while a single two-tube fixture was used in the (LD) photoperiod treatments. The photosynthetic photon flux density (PPFD, 400-700 nm), measured at the soil surface in the center of each band, averaged 75 pM m‘zsec"1 in the SD treatments and 35 PM m-Zsec-lmthe LD treatments. Overall, there was no significant difference in total daily quantum fluxes between the two photoperiodic treatments. The experiment was grown indoors, in a controlled temperature room. The temperature was maintained between 24 and 26°C and did not vary significantly between the two 100 photoperiods. The treatment combinations, (_i_._e_. photoperiod, size class, and cultural method (ageo) were arranged factorially in a split-plot randomized complete block design. Photoperiod formed the main plots. Cultural method, and size class formed the sub-plots. There were 3 main-plot replications with 6 sub-plot treatment combinations. Six seedlings of each treatment combination were randomly arranged within each main plot replication, for a total of 216 seedlings. Shoot growth phenology was recorded weekly, for 16 weeks. After budburst (when needles were first evident between the expanding bud scales), the shoot height from the bud base to the distal tip of the vertical most needle (flush height) was recorded for each seedling each week. Height growth rate differences among the treatments during the two different growth phases, flushing and subsequent shoot growth, were evaluated by comparisons linear regression slopes (Sokol and Rohlf, 1981). In order to assess gross morphology and development, samples were taken at five times during the experiment. The timing of the samples corresponded with observable changes in the shoot-growth phenology. Samples were taken: (1) of dormant stock, as received; (2) during budburst, at two weeks after planting; (3) at the end of preformed shoot elongation, four weeks later; (4) six weeks after that, during needle primordia initiation under short days; (5) and a last time, one month later, at the end of the experiment. 101 At each sampling time one seedling, of each of the 12 treatment combinations, was randomly selected from those remaining in each of the three main—plot replications. Subtending primordia were first counted, then the apical meristem and the upper few mm of the shoot were excised for microscopy, in a concomitant experiment. The rest of the main stem needles and needle primordia were then counted, diameter at cotyledon level recorded, and the shoots and roots separated. The seedlings were then oven-dried at 70°C and weighed. In order to test for total free—growth, needles were counted on the new formed shoots of the unsampled seedlings at the end of the experiment. These data were pooled with the data from the last sample, which was taken on the last day of the experiment. Free growth was determined as increases over means of SD shoot needle number in the LD seedlings of each corresponding sub-plot treatment combination. RESULTS Initial observations Within each size class, there was no initial difference in shoot height between the NUR and the AG seedling types. NUR seedlings did, however, have more mainstem needles per mm (113. higher plastochron index (PI)), greater stem diameter, and higher total dry weight than did the AG seedlings (Table 4.11. There were no significant initial 102 differences in root to shoot ratio. In addition to these quantitative differences, the shoots of the AG seedlings were morphologically different from the NUR seedlings, in ways not quantified. In general, the NUR seedlings had more lateral branches. These appeared to result mainly from the extension of lateral buds formed during the first year. The main stem buds of the NUR seedlings were larger, externally, than the AG buds. The AC needles were not as stiff as the NUR needles and were more normal to the stem axis (Figure 4.1). Table 4.1 Selected initial characteristics of commercial blue spruce nursery stock grown under conventional methods (NUR) for two years (2+0) and under accelerated growth conditions (AG) for one year. Type and Size Mean Plastochron Dry Root/Shoot Stem Class (mm) Height Index Weight Ratio Diameter 75-150 153a1/ 275a 0.73a2/ .64a 1.9a3/ AG 150-225 l9Sb 302a 1.00a .37a 2.6a 225-300 270C 37Gb 1.12a .45a 2.4a 75-150 139a 257a 0.83a .36a 2.5a NUR 150-225 200b 379b 2.10b .62a 4.3b 225-300 260C 488C 3.49c .65a 4.8b 1/ Means within a column, followed by the same letter, are not significantly different (p < .05) Duncan's Multiple Range Test. _ / Oven dry weights in grams. 3/ Stem diameters, at the cotyledon level, in mm. Figure 4.1 103 Representative blue spruce seedlings of the three size classes and two nursery culture methods, tested for subsequent free growth potential. 101+ hwy I; 'r H"! l|| Ill I'I Ill I'I [I I'I'li"~ hurl . IIIqu 1. ‘ I l o "J I ' IJ I I I 3 V up 3 O at. DU“ 'QLE. V . 105 The form of the NUR root system also differed from AG seedlings. The NUR root systems had fewer and coarser roots. They also had a more pronounced tap root than AG seedlings. The lateral roots of the AG seedlings were reflexed downward, about 90°, where they had encountered the wall of the container. On some AG seedlings there were indications of spiral roots forming. Also, the prominent tap root seen in the NUR seedlings was missing (Figure 4.1). Flushing and height growth There was no significant difference between the LD and SD treatments in the time to budburst, averaged over all seedling types. There was a difference, however, between the cultural methods. On the average, the AG seedlings broke bud and began shoot elongation within 1.5 weeks. The average for the NUR seedlings was 2.4 weeks. Within these two groups there were no differences in budburst date among size classes, nor were there any significant size by cultural-type interactions. Shoot elongation proceeded rapidly for all types of seedlings, under both photoperiods (Figure 4.2%. There was no significant difference in the average height-growth rate, during the first 6 weeks, between the LD and the SD treatments among size classes. There were, overall, significant height-growth rate differences between the NUR and the AG seedlings during this phase (p=.001). NUR seedlings average height-growth rate was 42% greater. Preformed shoot growth, under short days, was complete by Figure 4.2 106 New flush heights for AG and NUR seedlings grown for 16 weeks under two photoperiods. A) Cumulative flush height curves for three size classes of AG blue spruce seedlings grown under 24 hr photoperiods. B) Cumulative»flush heights curves for three size classes of AG blue spruce seedlings grown under 12 hr photoperiods. (D Cumulative flush height curves for three size classes of NUR blue spruce seedlings grown under 24 hr photoperiods. In Cumulative flush height curves for three size classes of NUR blue spruce seedlings grown under 12 hr photoperiods. SEEDLIND FLUBN NEIDNT (NH) 100 00 00 70 00 50 40 30 20 10 100 90 00 70 60 50 40 30 20 10 - — 00 78-150 NH ""” ND 160-225 NN "" 60 225-300 NH 'IIIIIIIIIIIIIII - "'"' NUR 75-150 PM I‘\ _" NUR 150-225 H11 ---- NUR 226-300 np/ ' ,1”7*--~ .52: I, ’r” ‘ I A I ”’ \\-"’” l ‘ QT ‘§~- 1» ° '1 IIIIIIIIIIIIII 10 12 14 180 107 A — 90 75-150 NH -‘ """ 90 150-225 NH "" 90 225-300 NH ooooo o o ’0' 'l I 'l I .. IrlllTlflTllllI l --- NUR 75-150 an —- nun 150-225 m1 ---- NUR 225-300 rm h———’—— \ J '0 \ "~ ....... ‘ q /0 ‘ s‘ "/ I”\\ ----- \ ‘b ...... 0 2 4 0 0 NEEKB DE DRDNTH UNDER L0 IIIIIIIIIIIIIII 2 4 6 810121416 NEEKS 0F 0RONTN UNDER 50 108 the sixth week of the experiment. By this time, the average NUR seedling flush height was 28% greater than that of the AG seedlings (Figure 4.2%. Post flushing growth Under SD, al 1 seedlings had set dormant buds by the end of shoot elongation. There was no overall change in shoot height during the last 10 weeks of the experiment for any of the NUR or AG SD seedlings (Figure 4.2Bfin. There was significant shoot height-growth, over the last 10 weeks of the experiment, for both AG and NUR seedlings, under LD. AG seedlings grew 75% faster than the NUR seedlings, averaged over size classes, during this period. Under LD all AG seedlings grew at the same rate. There were significant differences among size classes within the LD NUR seedlings. While the smaller two size classes, 75-150 and 150-225 mm, did not differ, their average growth rate was 67% greater than the 225-300 mm size class (Figure 4.2A,C). Total seedling dry weight also increased throughout the experiment for both NUR and AG seedlings. There was no overall photoperiodic effect on total weight increase (Figure 4.3A). In contrast to total dry weight, there were significant differences in the root to shoot ratio between the photoperiods. LD seedlings had significantly lower root to shoot ratios than SD seedlings (.32 vs. .36, p=.05). Overall, the AG seedlings had higher root to shoot ratios Figure 4.3 109 Biomass accumulation and distribution in NUR and AG blue spruce seedlings grown for 16 weeks under 12 hr and 24 hr photoperiods. A) Total seedling dry weight for AG and NUR seedlings averaged over size class and photoperiod. B) Root to shoot ratios of AG and NUR seedlings averaged over size class for both 12 hr and 24 hr photoperiods. SEEDLING DRY WEIGHT (on) Root/swear “no Vir1I1—IIU'_I—1 456789IO|IIZI3I4|5|6 WEEKS OF GROWTH —1 NJ (flu! NUR SD NUR LD ——— AG 50 --—- AG Lo l2 456789l0l|l2l3l4l5|6 WEEKS OF GROWTH 111 than the NUR seedlings. There were also differences in root to shoot ratio among the sample times. Root to shoot ratio was highest among the seedlings prior to their potting at the beginning of the experiment. It declined overall until the end of shoot elongation then began to increase under SD but not under [.0 (Figure 4.38). Final observations After 16 weeks of growth under LD, all size classes of AG seedlings had significantly more needles than did their SD counterparts. On the average, 50% of the AG LD flush needles were neoformed or "free growth". There were also Significant differences in the final flush-height, between photoperiods, in the AG seedlings. LD flush height averaged 30% greater than SD flush height. Under LD there was less, but still significant, free growth in the NUR seedlings, as well. NUR seedling shoots, on the average, had produced only 22% of their needles by free growth by the end of the experiment. The NUR free growth was highest on the 225-300 mm seedlings on which 32% of the flush needles were formed by free growth. Although the means of all the LD NUR treatments had more stem units than the corresponding SD treatments, this was the only individual treatment combination which showed significant free growth. In contrast to the AG seedlings, there was no significant flush height difference between LD and SD for the last sampled NUR seedlings, due to the extremely short internodes of the free growth stem units. 112 Table 4.2 Selected characteristics of 2-year-old blue spruce nursery stock (NUR) and one-year-old, accelerated-growth (AG) stock after flushing and growth for 16 weeks under 12 hr (SD) and 24 hr (LD) photoperiods. Type and Size Flushl/ Plastochronz/ Dry Root/Shoot Stem Class (mm) Height Index Weight Ratio Diameter ————————————————————————————— LD -..-..-...._..--_____--_.._--..--_.. 75-150 93 *2/ 225 * 1.35 aiié/ .41 cd 2.9 _5/ AG 150-225 86 * 210 * 2.00 abc .34 abc 2.8 a 225-300 82 * 193 * 2.79 abc .32 abc 3.0 ab "m’333126""$3'I""113’1 5° "CBS"2.8""?12'5“'"3T§‘;‘ AG 150-225 63 * 106 * 1.71 ab .41 cd 2.8 a 225-300 61 * 97 * 3.04 be .30 ab 3.7 bc Ii ___________________________ LD ____________________________ 75-150 81 ns. 257 ns. 1.67 ab .27 ab 2.7 a NUR 150-225 99 * 284 * 3.61 cd .25 a 3.8 bc 225-300 84 ns. 271 ns. 4.64 def .25 a 4.0 c """ §§II§6""ES';QT"SES'ES§D 7372;""TSE'SEJMSTS'QQ NUR 150-225 86 * 190 * 4.14 cde .33 abc 4.1 c 225-300 79 ns. 211 ns. 5.67 f .31 abc 4.4 c £77He1ght of the new formed terminal shoot in mm. 2/ Number of needles and needle primordia on the new shoot. 3/ Means followed by *, within a type and size class, are significantly different (p=.05, LSD) between photoperiods. 4/ Means within a column, followed by a common letter, are not significantly different (p < .05) Duncan's Multiple Range Test. - / Oven dry weights in grams. 6/ Stem diameters, at the cotyledon level, in mm. 113 By the last sample, the average dry weight was 82% greater than the initial value. The AC seedlings increased dry weight by 2.18 times while the NUR seedlings increase was only 69% of the initial value. There was no significant photoperiodic effect on final dry weight (Table 4.2). The root to shoot ratio was greater, in the SD seedlings than in the LD seedlings at the last sample time. There were still significant differences among the size classes. Overall, the tallest seedlings had the lowest root to shoot ratios. The stem diameter had also increased significantly during the experiment. ‘While the NUR seedlings had the greatest stem diameters at all sample times, diameter growth was only significant among the AG seedlings. Photoperiod had Ix) effect on stem diameter growth (Table 4.2). DISCUSSION The AG seedlings used in this investigation initially differed quantitatively and qualitatively from the similar sized NUR seedlings used. Therefore, age was not the only difference between cultural treatments among size classes. From the responses observed, it seems likely that some of these differences had a strong effect on the free growth propensity of these seedlings. The earlier budburst in the AG seedlings was most likely due to the more advanced phenology of their buds, as they were received. This was investigated in a concomitant 114 experiment (Chapter 5). Differences in the bud phenology may be due to the winter storage environment. Photoperiod and temperature affect bud phenology, in cold stored conifer seedlings (Arronson, 1975; Lavender, 1980), and they were not known. Since the NUR seedlings were stored bare rooted and the AG seedlings had a "plug" of moist growing medium surrounding their roots, the storage environment may have affected the two classes differently. That the average height-growth rate during the flush phase was greater for the NUR seedlings than the AG seedlings and was not significantly different between the LD and SD treatments is not surprising. Temperature, rather than photoperiod, is generally regarded as the most important environmental stimulus of budburst, of a wide variety of woody plants (Kramer and Kozlowski, 1979). This effect of temperature may be cumulative, as seems to be the case in mature white spruce (Owens gt 31,, 1977). Since the NUR seedlings had more preformed needles in their buds, they would be expected to have a greater flush growth rate. If there was an equal rate of internode extension, flush growth rate should be directly proportional to the number of preformed stem units within the bud. The internode extension was not, however, uniform. While the NUR buds had nearly twice as many needle primordia as those of the AG seedlings, the average height-growth rate for the NUR seedlings was only 72% greater than the AG seedlings. In established spruce trees, the number of 1E5 preformed primordia accounts for up to 92% of the variation in shoot growth rate, within a given site (Cannell 35 31., 1976). The loss of internode elongation "vigor" in the NUR seedlings may have resulted from a number of external factors rather than any endogenous change in shoot growth- rate potential. The reduction in the following year's growth, after transplanting, in bare-rooted nursery stock (planting check, (Sutton and Tinus, 1983)) is commonly observed in spruce (Burdett st 21,, 1984). Burdett and others (1984) also found that plug seedlings of Engelmann and white spruce, similar in size to those used in this investigation, suffered less from transplanting than bare root nursery stock. The combined effects of root damage, during lifting and storage, may have lowered the potential stem growth rate by lowering the seedling water potential during the shoot extension phase. In field planted ponderosa and lodgepole pine (Pinus ponderosa Laws. and 3. contorta DougJ, transplanting effects can lower the water potential for at least three years after planting (Baldwin and Barney, 1976). Root pruning lowers spring water potential and shoot elongation, in older blue spruce, in the field (Marquard, 1983). Photoperiod had no apparent effect on internode or needle elongation, during the flushing phase. This implies that the the meristematic tissues involved in the final 116 development of these organs, 3.3. within the preformed stem internodes and needle primordia, may not be governed by the photoperiod. If they are, it is not as distinct as the photoperiodic control of organ differentiation and development, at the shoot apical meristem, under free growth conditions (33. Cannell, 1978b). Throughout the experiment, a 12 hr photoperiod was assumed to induce no free growth. Since no significant increase in the number of needles on the developing shoot was observed at any sample time under SD, the number of needles on the short day shoot was considered to be the same as that in the bud (Pollard and Logan, 1977). In most studies involving spruce germinant seedlings, shoot growth stopped under 12 hr photoperiods well before seedlings were as old or big as any used in this investigation (3.51. Heide, 1974a; Pollard gg‘gl., 1975; Young and Hanover, 1976). Shoot growth continued or resumed under LD, after shoot elongation, in all seedling types. However, the rate of shoot growth was much lower than that observed previously in germinant and young seedlings of blue spruce, grown under similar conditions (3.3. Young and Hanover, 1976, 1977b, 1978; Chapter 1). Here, as with the flush growth phase, the internode distance was less in the NUR seedlings. In the AG LD material in this study, the average height growth rate after the elongation of the preformed shoot was 2.9 mm/week while for the NUR LD seedlings it was 1.7 mm/week. The reduction in root to shoot ratio during the early 117 part of the experiment may have arisen from at least two processes. The loss may have been due to the translocation of storage materials, from the roots, to the shoot during budburst and flushing (Kramer and Kozlowski, 1979). A second possibility is that the relative weight loss was caused by the preferential allocation of new photosynthate to the shoot (Loach and Little, 1973). Since there was some dry weight increase of the roots by last sample, in most treatment combinations, both may have occurred but at different rates during the experiment. The higher root to shoot ratios seen under SD, overall, probably arose in a similar manner. Shoot growth stopped at the end of shoot elongation in these seedlings and relatively'more photosynthate could then have been available for translocation to the roots. The principle reasons for the low growth rates seen in this experiment appear to be transplanting shock and low PPFD. The former reason is substantiated, but not proved, by both the lowered flush vigor and lower weight increase in the NUR seedlings, which had received far more mechanical disruption of their roots, in their previous culture, than the plug AG seedlings. That PPFD was also limiting is suggested by the comparison of the average shoot height-growth rates and the PPFDs of the germinant seedlings grown for the experiment in Chapter 1 with those of the AG LD seedlings here (which had received relatively little root disturbance). Both the 1H3 average height-growth rate and PPFD were about three times greater for seedlings in that experiment than those seen in this investigation. The average light intensity used under LD in this investigation was less than 10% of the photosynthetic saturation value for blue spruce seedlings: which is less than that needed for optimum growth (Tinus and McDonald, 1979). In conclusion, seedling heights within the range of 75 to 300 mm seem to have no effect on the ability of spruce seedlings to free grow, in the first two years. In older seedlings, the size and age may, or may not, have independent or collective effects on the ability to free grow. In order to objectively evaluate this phenomenon, in light of the long term effects of root disturbance on seedling shoot growth and physiology seen here and elsewhere (Baldwin and Barney, 1976; Burdett st 31, 1984), further comparisons should be made on undisturbed material grown either entirely in pots or directly in the ground. CHAPTER 5 THE EFFECTS OF PHOTOPERIOD ON SHOOT APICAL DEVELOPMENT, ANATOMY, AND ULTRASTRUCTURE IN BLUE SPRUCE SEEDLINGS. ABSTRACT The shoot apical development of three size classes of blue spruce (Picea pungens Engelnn) seedlings, grown under long and short days was studied. The seedlings had previously been grown for either two years (2+0) under conventional nursery practices (NUR) or less than one year under accelerated-growth greenhouse culture UKD. All seedlings grown under the short day (SD) treatment set buds at the end of shoot elongation. Under long days (LD), the response was less clear. Most seedlings of all types resumed "free growth" under LD treatment. Some nursery grown seedlings set buds which did not break during the course of the experiment. Normal bud development followed bud set in all cases under short day treatments. After 16, and in many cases by 12 weeks, the apical meristem of the SD treatment seedlings lost.a‘well defined cytohistological zonation and cell walls appeared shrunken. In addition, the cells of of all zones of these meristems had smaller vacuoles and more starch grains. These changes were associated with the development of a normal preformed shoot within the resting bud. 1E) 120 Under long days, those seedlings which resumed free growth had apical architecture similar to those previously observed in spruce germinant seedlings, grown under long days, that had fully developed apical meristems. Zonation was well developed and primordia formed from the peripheral zone tissue developed directly into needles. In contrast to developing germinant seedling meristems, and perhaps due to low photosynthetic photon flux density levels, total meristem volume decreased over time. Those seedlings setting bud and not resuming free growth under long days had large conical meristems similar to those seen in naturally grown material late in the bud scale formation period. Prolonged bud scale formation seems to occur under LD in seedlings not exhibiting free growth. This suggests a change, rather than a loss, of photoperiodic response as in older spruce seedlings. 121 I NTRODUCT I ON The annual cycle of shoot growth and bud development in mature spruce (Picea _p. (An) Dietr.) trees, growing under natural conditions, has been well described for a number of species (Fraser, 1962, 1966; Owens and Molder, 1976; Owens, 35 31., 1977; Pillia and Chacko, 1978; Thompsett, 1978; Harrison and Owens, 1983). In germinant seedling and young seedlings, grown under natural conditions, the determinate growth pattern seen in mature trees is modified by the inclusion of photoperiodically induced, indeterminate growth (Jablanski, 1971; Powell, 1982). While few anatomical studies of bud development and phenology have been made on young spruce seedlings (Burley, 1966b; Gregory and Romberger, 1972; Cannell, 1978a), considerable work has been done on the environmental control of bud development in germinant and older seedlings (3.3. Fraser, 1966; Dormling: ‘gtlgl., 1968; Pollard, 1973; Heide, l974a; Pollard and Logan, l974b,l977; Young and Hanover, 1977b; Cannell and Cahalan, 1979; Pollard and Logan, 1979). No previous investigations have»compared controlled-environment effects on spruce apical meristem anatomy and ultrastructure. Spruce seedlings loose the propensity for indeterminate or "free" growth as they age. This loss includes both the free growth response under natural photoperiods and the response to artificially extended photoperiods. In blue 122 spruce, for example, germinant and one-year-old seedlings grow indeterminately under 24 hr photoperiods. Two year old seedlings appear to be at a transitional stage, some resuming free growth and others not (Young and Hanover, 1976). While three-year-old and older blue spruce will not grow indeterminately, spruce that has passed through more than three artificially shortened growth cycles will (Chapter 3). The free growth phenomenon, therefore, does not seem to be linked, specifically, to the cyclic changes in meristematic activity and bud development seen during the first three years. Since blue spruce seedlings can be grown, under optimum conditions, larger than most two-year-old seedlings (_e_._g. Hanover and Reicosky, 1972; Chapter 1), shoot size peg s3 does not appear to be a limiting attribute either. Blue spruce seedlings grown under accelerated-growth (AG) conditions, using extended photoperiods, for several months then allowed to form buds prior to chilling all resumed free growth when returned to warm temperatures under long days. Two-year-old (2+0), conventional nursery-grown seedlings (NUR), of the same shoot height classes, were intermediate in their response. Some NUR seedlings appeared to resume free-growth. Other seedlings formed resting buds and never resumed shoot growth. No clear relationship was found between the reinitiation of free growth and any single, measured characteristic of the NUR seedlings, 123 although root damage in nursery handling was suggested (Chapter 4). The objectives of this experiment were to determine if there were photoperiodically induced differences in bud development and anatomy between similar sized AG and NUR blue spruce seedlings. Also, it was hOped to gain further understanding into the role of the photoperiod in the development of dormant buds in a uniform environment. In addition, an attempt was made to determine if there were developmental, anatomical, or ultrastructural differences in the apical meristems, associated with the presence or absence of free growth, under 24 hr photoperiod. MATERIALS AND METHODS The apical regions of blue spruce seedlings, of the age, size and cultural treatments sampled for the investigations of Chapter 4 were used in this experiment. Two photoperiods were used, long days (LD), 24 hr of continuous irradiation and short days (SD) of 12 hr of irradiation at twice the daily quantum flux as the LD treatments (see Chapter 4). Five samples with three replications from each of the treatment combinations were taken throughout the experiment. These sample times, corresponding with gross phenological changes in the seedlings, were: (1) dormant buds as the seedlings were received; (2) during budburst two weeks after 124 planting; (3) at the end of preformed-shoot elongation; (4) at the end of bud-scale initiation (as seen under short day treatments); and (5) late needle initiation (also short days), after 16 weeks of growth. At each sample time, the main-stem apical meristem and any preformed, unexpanded primordia associated with it were excised and plunged immediately in to ice-cold, PIPES buffered (Piperazine—N-Ntébis (2-ethane sulfonic acid)), glutaraldehyde/paraformaldehyde fixing solution OCecich, 1977). When the apical meristem and preformed shoot were covered by bud scales, most of these were removed first. The tissue was then exhausted under vacuum and allowed to fix overnight under refrigeration (4°C). After five 10 to 15— min washes in plain PIPES buffer, the material was post fixed with osmium tetroxide as described by Cecich (1977). The material was then dehydrated in an acetone:water series with 25% concentration steps, changed hourly. After two additional changes of 100% acetone, the last one overnight, the apices were infiltrated, with agitation, with Spurr's medium-hard resin (Spurr, 1969). A similarly graded acetone:resin series with 24 hr between steps was used. The specimens were finally given three changes of 100% resin. Following the last resin change, the specimens were cast in blocks and the resin polymerized for about 72 hr at 60°C. For light microscopy, the apices were sectioned, longitudinally, up to the median plane at 2 pm thickness on a Porter-Blum ultramicrotome using a glass knife. By 125 monitoring sectioning progress every 5 to 10 sections the, median plane could be detected to within one cell thickness of dead center. The sections were mounted on glass slides dried, then stained with methylene blue-azure A with or without basic fuschin counterstain (Berlyn and Miksche, 1976). After drying, cover slips were affixed with Permount. The same photographic and digitizing techniques used in Chapter 2 were then used in quantifying apical meristem development. In addition to the volumetric data, relative zonation was scored on a scale of 0 to 3, based on increasing clarity of cytohistological zonation. Selected apices were also sampled for transmission electron microscopy (TEM). For TEM pale gold (90 nm) and thinner sections were made with a diamond knife. These sections were mounted on copper mesh grids and stained with uranyl acetate (Stempak and Ward, 1964) and lead citrate (Reynolds, 1963). The lead procedure was modified by the addition of barium chloride, to precipitate carbonate, to the washing solutions. These grids were then viewed in a Philips TM 300 or a TM 201 transmission electron microscope operating at 80 or 60 kV respectively. Micrographs of selected areas at various magnifications were made on Kodak 4489 electron imaging film. At both the initial and final samples, apical regions from representative seedlings were also viewed with a 126 scanning electron microscope (SEM). These samples were mounted on aluminum stubs with graphite cement and viewed fresh, in an ISI Super III scanning electron microscope, operating at 15 kV. RESULTS Initial observations Buds of the NUR seedlings at the initial sample had more than twice the performed preformed needle primordia in their buds than the AG seedlings (Table 5.1, Figure 5ND. There were no significant differences among size classes within the two nursery culture techniques. The NUR buds were also larger in external appearance. Table 5.1 Selected initial characteristics of the terminal buds of commercial blue spruce nursery stock grown under conventional methods (NUR) for two years (2+0) and under containerized, accelerated-growth conditions (AG) for one year. Cultural Preformedl/ Meristemg/ Height Diameter Method Needles Volume (pm) (pm) AG 1022/ 2891 103 276 NUR 203 6780 136 350 I] NeedIes elongated under SD treatments. 2/ In pm3 x 103, calculated from median longitudinal sections. 3/ All means, between cultural methods, were significantly different (p£.05). Clear phenological differences were also apparent between the AG and the NUR seedlings at the initial sample. 127 The AC seedling buds had progressed further towards budbreak than those of the NUR seedlings (Figure 5.1). The start of vascular differentiation in the proximal cortex of the preformed shoot had progressed acropetally to nearly the top of the expanding preformed shoot. Needles of the AG buds were further developed, completely over-arching the apical meristem (Figure 5.1A). Most of the buds of the NUR seedlings, while not as far advanced as those of the AG seedlings had started to elongate (Figure 5.1B,E,F). Both needle elongation and vascular development had started in these as well. There were also anatomical differences in the apical meristems of the seedlings of the two cultural methods, at the initial sample. The apical meristems of the AG seedlings were smaller, overall, than those of the NUR seedlings. This difference was due to both increased height and diameter of the NUR seedlings (Table 5.1). The meristems of the less advanced buds were anatomically different from those in buds further advanced towards bud break. These differences were observable at light-microscopy resolution and at the TEM level. The most obvious difference between the less and further advanced apical meristems was the shrunken appearance of the cells within the less advanced meristems. This shrinkage, visible at the light microscopy level, did not involve the separation of the plasmalemma from the cell wall (Figure 5.2). As seen in Figure 5.2A, the shrinkage Figure 5.1 128 Preformed shoots from buds of AG and NUR blue spruce seedlings. A) B) C) D) E) F) Scanning electron micrographs of the preformed shoot in an AG seedling terminal bud. Bar = 1 mm. Median longitudinal sections through the. preformed shoot in an AG bud. Embedded in epoxy and sectioned at 2 pm. Bar = 500 um. Median longitudinal section through the shoot apical meristem in the bud of an AG seedling. Bar'= 100 pm. Scanning electron micrographs of the preformed shoot in a NUR seedling terminal bud. Bar = 1 mm. Median longitudinal sections through the. preformed shoot in a NUR bud. Embedded in epoxy and sectioned at 2 pm. Bar = 500 pm. Median longitudinal section through the shoot apical meristem in the bud of a NUR seedling. Bar = 100 pm. .\. . “W... . _. .. .5..- owl. x v 41.5.sz 7 . Figure 5.2 130 Light and transmission electron micrographs of the shoot apical meristem of a dormant blue spruce seedling, prior to the start of elongation. A) Light micrograph of a median longitudinal section of the apical meristem. Note low contrast of the nuclei and shrunken appearance of cells. Bar = 75 pm. This condition existed through out the preformed shoot, note last formed needle primordium (np). B) Higher magnification light micrograph of the central mother-cell zone area of the same apical meristem. Note small vacuoles and high concentration of refractile starch grains. Bar = 15 pm. C) Transmission electron micrograph of an apical- initial central mother-cell zone cell. Note convoluted cell walls, heterochromatin Gun in the nucleus and starch free plastids (p). Bar = 2 pm. D and E) Central mother-cell zone cells showing contorted cell walls, numerous starch filled plastids and osmiophillic (lipid) bodies. The vacuoles (v) present in the these cells often had membranous inclusions or infoldings. Bar = 2 pm. F) Plastids and inclusions in the central mother- cells. The plastids had large starch grains (5) and little lammelar development. Lipid bodies (1b) were common. They were not seen to be associated with endoplasmic reticulum cisternae but but were observed in association with some vacuoles. The dense cytoplasm in these cells appeared to be due to large numbers of ribosomes (r). Bar = 500 nm. 132 was not restricted to any area of the meristem. The degree of shrinkage appears uniform throughout the meristem and subtending needle primordia, but did not appear to extend into the sub-crown cortex or pith. In all meristems, the central mother-cell zone appeared to have more vacuoles then the surrounding tissues. The vacuoles were smaller in the less advanced meristems than those observed in typical active meristems. In addition, the membranes of many of the vacuoles appeared to have lipid inclusions or associations and infolded membranes, conditions not seen in the vacuoles of active meristems (S.ZD,E). The cells of the less advanced meristems, especially those of and near the central mother-cell zone, were richer in two types of "storage" compounds. The plastids of these cells had more and larger starch grains (Figure 5.2). In addition, they contained more osmiophillic (presumptively lipid) bodies. These bodies ranged in size up to about 1 um in cross section (Figure 5.2F). They did not appear to be membrane bound nor to have any association with endoplasmic reticula. At the light-microscopy level, these bodies appeared to be non-birefringent and non-refractile. Starch grains were non-birefringent but refractile, depending on the plane of focus (Figure 5.1A,B). These small starch grains also stained pink with basic fuschin as did the large grains seen later in the cells of the developing pith of the SD buds. Both types of bodies were nearly absent in the 133 further developed meristems (cf. Figure 5.1C,F and Figure 5.2A,B). Lastly, the nuclei of the central mother-cell zone of the less advanced meristems were more heterochromatic than the more advanced ones. The lower contrast between the nuclei and the surrounding cytoplasm in the less advanced meristems, seen at both the light microscopy and TEM levels appeared to be due mainly to a higher concentration of ribosomes in the cytoplasm of these cells (Figure 5.2f). Bud break and shoot elongation Bud-break was significantly earlier in the AG seedlings. The AG buds broke, on the average, in 1.5 weeks while the NUR seedlings took 2.4 weeks. There were no significant differences among size classes within cultural treatments. In addition, there appeared to be no photoperiodic influence on time to bud break (Chapter 4). The volume of the apical meristems, averaged over all seedling sizes and nursery treatments, declined throughout the experiment. There were no overall differences in meristem volume among the different seedling size classes within the nursery culture techniques nor were there any significant differences between the NUR and AG meristems averaged over the course of the whole experiment. There were, however, three significant interactions. The meristems of the NUR seedlings, averaged overall sample times did not differ significantly between the LD and SD treatments while the AG seedlings did. The average SD, 134 NUR meristem volume was 4.67 x 106 pm3 while under LD it was 4.33 x 106 pm3. For the AG seedlings the corresponding means were 5.00 x 106 and 2.812 x 106 pm3, for the SD and LD treatments respectively. The second differential response was that of the two photoperiod averages at the four sample times. Under LD, there was an overall decline in meristem volume while under SD there was an overall increase followed by a decline. The greatest differential response was that between the two nursery treatments and photoperiods at different sample times (Figure 5.3). Its significance results from the later and greater increase in meristem volume seen in the AG seedlings under SD. There were significant differences in the distinctness of the cytohistological zonation between the two photoperiods. The meristems of all types had more distinct zonation under LD. The distinctness of the zonation declined over the course of the experiment after peaking at the second sample time. In addition to the quantitative changes in apical meristem volume and zonation, there were changes in the types of derivatives of the meristems. These differed with photoperiod and sample time and are related to the quantitative changes described above. At the first sample, the last formed primordia of all types of seedlings appeared to be needle primordia (Figure 3.1C,E). By the second sample, during bud burst, most of the meristems appeared to 135 Figure 5.3 Average total apical meristem volumes for AC and NUR blue spruce seedlings, pooled over heights, grown under LD and SD photoperiods for 16 weeks. (um3 x IOS) MERISTEM VOLUME 136 ’ NUR so \’ NUR LO AG SO AG LD (flu: U I U I I I I I I I I 4 5 6 7 8 9 IOII l2l3l4|5l6 WEEKS OF GROWTH 137 have primordia differentiating into bud scales on their flanks. This was true under both LD and SD treatments and for all sizes of NUR and AG seedlings. At the end of shoot elongation under SD, photoperiodic differences first became apparent. Under long days, apical activity followed two different routes. .After differentiating several bud scale primordia, many meristems returned to the production of needle primordia which subsequently developed directly into needles. This was visible by the third sample time. Some meristems, all of the NUR nursery treatments, differentiated only bud scales. Final observations By the end of the experiment, after 16 weeks of growth under the two photoperiodic treatments, major differences were evident among the apical meristems and developing primordia of the 12 treatment combinations. All seedlings grown under the SD treatment had well developed buds. Under LD, two types of apical development were observed. Seedlings under long days were either free growing and had not recently formed bud scales (FGLD), or had formed visible buds and were not free growing (NGLD) The SD buds of all size classes of NUR and AG seedlings had developed morphologically complete performed shoots (Figures 5.4 and 5.5). In all cases, the needle primordia were less developed and shorter than those in the buds at the beginning of the experiment. No vascular development Figure 5.4 138 Apical regions of AG blue spruce seedlings after flushing and growth under 12 hr and 24 hr photoperiods. A) Scanning electron micrograph of the apical region of an AG seedling grown under LD. Bar = 500 pm. B) Median longitudinal section through the apical region of an AC seedling grown under LD. Bar = 500 pm. C) Median longitudinal section through the shoot apical meristem of an AG seedling grown under LD. Note the large vacuoles and large lightly staining nuclei in the central mother-cell. Bar = 100 pm. D) Scanning electron micrographs of the apical region of an AG seedling grown under SD. All but two bud scales have been removed. Bar = 500 pm. E) Median longitudinal section through the apical region of an AC seedling grown under SD. Note crown formation at base of preformed shoot. Bar = 500 um. F) Median longitudinal section through the shoot apical meristem of an AG seedling grown under SD. Note the less distinct zonation, shrunken cells, smaller vacuoles and abundant dense inclusions. Bar = 100 pm. 140 Figure’5.5 Apical regions of NUR blue spruce seedlings after flushing and growth under 12 hr and 24 hr photoperiods. A) Scanning electron micrograph of the apical region of an NUR seedling grown under LD. Bar = 500 pm. B) Median longitudinal section through the apical region of an NUR seedling grown under LD. Bar = 500 pm. C) Median longitudinal section through the shoot apical meristem of an NUR seedling grown under LD. Note the large vacuoles and large lightly staining nuclei in the central mother-cell. Bar = 100 pm. D) Scanning electron micrographs of the apical region of an NUR seedling grown under SD. All but two bud scales have been removed. Bar = 500 pm. E) Median longitudinal section through the apical region of an NUR seedling grown under SD. Note crown formation at base of preformed shoot. Bar = 500 um. F) Median longitudinal section through the shoot apical meristem of an NUR seedling grown under SD. Note the less distinct zonation, shrunken cells, smaller vacuoles and abundant dense inclusions. Bar = 100 pm. 142 was evident in the cortical regions of the preformed shoots. Although counts of total needle primordia were not made, the buds of the AG seedlings appeared to have more primordia than those of the NUR seedlings (33. Figures 5.4D5E and 5.5D,EL. In median longitudinal section, the buds of the AG seedlings had 36% more needle primordia than the NUR bud preparations. At the base of all SD buds, just proximal to the bud scale receptacle, a well developed crown region had formed (Figures 5.4E and 5.5E). The pith cells immediately proximal to the crowns were large and lightly staining but had not broken down. Thus, no cavity had formed. The cytohistological zonation in the SD apical meristems was less distinct than that of the FGLD meristems. At the light microscopy level, the nuclei stained much less distinctly and the cytoplasm much more darkly. These factors, combined with the fewer and smaller vacuoles reduced the overall within and between cell contrast, compared to the FGLD meristems (Figure 5.4C, 5.5C). In the central mother-cell zone and upper pith-rib meristem areas, concentrations of starch grains were seen. ‘Vacuoles in the apical initials and central mother-cell zone were small and less numerous then in the FGLD meristems. The cell walls of the SD meristems appeared wrinkled. At the TEM level of resolution, further differences were apparent. The walls of the cells bounding the SD meristems were wrinkled, but the sunken appearance, with 143 the walls protruding only at the junction with adjoining cell walls (Sf. Figure 5.2C and Figure 5.6A), was not generally apparent. This type of cell wall distortion was seen throughout the SD meristems, and their subtending primordial. shoots (Figure 5~4F and Figure 5.5E). In the central mother-cell zone of the SD meristems, the vacuoles were small, often with infolded membranes. The plastids had starch grains, but the grains did not appear to be quite as big as those seen in the less advanced initial samples (sf. Figures 5.2D and 5.6D). Lipid bodies, also most obvious in the central mother-cell zone, were more abundant in the SD meristems. They also appeared to be smaller than those seen in the less advance meristems of the initial sample. The nuclei of the central mother-cell zone were more heterochromatic than those seen under the FGLD seedlings. They appeared, however, to lack the large, contiguous aggregations of heterochromatin seen in the less advanced initial material. Heterochromatic nuclei of similar description were seen in the pith-rib meristem and the peripheral zone (Figure 5.6E). Again, as with the initial samples, the darker staining of the cytoplasm was largely due to a high density of ribosomes. Under LD, two types of apical development and activity were seen by the end of the experiment. The apical meristems of most LD seedlings appeared to be free growing (FGLD) (Figure 5.4A, 5.5A). Some meristems, all from the NUR seedlings, had formed only bud scales under LD and were not 144 free growing (NGLD), even after 16 weeks (Figure 5.7A,B). The FGLD seedling meristems differed qualitatively and quantitatively from the SD. The average meristematic volumes were significantly smaller (Figure 5;”. The FGLD meristems also had more clearly defined cytohistological zonation than did the SD meristems. Meristematic cell turgor also appeared to be greater in FGLD seedlings, as the cell walls of these apical meristems were less wrinkled than those of the SD meristems (Figures 5.4, 5.5, 5.7). This was especially evident along the free margin of these meristems (Figure 5.7 A,D,E). The cytohistological zonation, at the light microscopy level, in the FGLD meristems was largely the result of the differential distribution of vacuoles, differences in nuclear size and staining, the contrast between the nuclei and the cytoplasm, and the alignment of the cell walls. These observations were also related to ultrastructural characteristics. In general, the differences in cell contents among cytohistological zones were greater within the meristems of FGLD seedlings than within the SD seedlings (gfi. Figures 5.6, 5.7). The FGLD central mother-cell zone cells were lightly staining. On an ultrastructural level this appeared to be due to more and larger vacuoles, large euchromatic nuclei and a lower density of ribosomes in the cytoplasm (Figure SIN. The vacuoles in the central mother cell zones of free-growing meristems often comprised the major part of the Figure 5.6 145 Transmission electron micrographs of cells of the shoot apical meristem of a representative AG seedling grown under SD. A) Apical initial cells showing contorted cell walls (w) and some shrinkage along the upper meristem boundary (mb). Bar = 1 mm. B) Central mother-cell zone. Note contorted cell walls and starch grains forming in plastids. Bar = 1 pm. C) Plastid (p) in central mother-cell zone. Starch grain is relatively small and has a distinct lammelar structure surrounding it. The plastids generally appeared less electron lucent than mitochondria (m) which were about the same size. Bar= 500 nm. D) Central mother cell zone showing plastids with larger starch grains (5) and numerous lipid bodies. The nuclei of these cells had some heterochromatin (hc) evident. Bar = 2 pm. E) Peripheral zone cells. These cells had large numbers of ribosomes in their cytoplasm yielding the dark image. Cell walls were perforated by numerous plasmodesmata (p), as were those of the other zones. Cell walls were also contorted in this zone. Bar = 5 pm. F) Rib—pith zone cells. Some cells of this region had large tannin filled vacuoles (tv). The plastids of the rib-pith meristem also had large starch grains in many of them. The nuclei in this zone were highly heterochromatic. Bar = 1 pm. Figure 5.7 147 Transmission electron micrographs of cells of the shoot apical meristem of a representative NUR seedling that showed free growth under LD. A) Apical initial cells showing turgid cell walls and large vacuoles (v) without membranous inclusions. Bar = 3 pm. B) Central mother-cell zone. Note contortion free cell walls and large euchromatic nucleus (en) Bar = 3 pm. C) Central mother-cell zone cell. Some heterochromatin was evident in some central mother-cell zone cells. The cytoplasm is less densely staining apparently from a lower concentration of ribosomes. Endoplasmic reticula (er) was more easily seen in these cells. While most of the central mother-cell zone cells had large nuclei, some had a number of smaller separate vacuoles. Bar= 2 pm. D) Peripheral zone cells showing plastids without starch grains. The vacuoles in the peripheral zone cells were smaller than those in other regions. turgid cell walls were apparent throughout this region. The nuclei of these cells were highly heterochromatic (hn) with prominent nucleoli (n). Bar = 4 pm. E) Peripheral zone cells showing telophase mitotic figure. The cytoplasm of the peripheral zone appeared to be richer in ribosomes than that of the central mother-cell zone. Bar = 4 pm. F) Rib-pith meristem cells. The plastids of the rib-pith meristem had large starch grains in many of them. The nuclei in this zone were highly heterochromatic. Most of the cells had numerous small vacuoles. Bar = 4 pm. ..... ,, , .v/J ’ t. ,5 . Aw . 149 cell. There were other ultrastructural differences seen in the apical-initial central mother-cell zone as well. Fewer plastids were seen in the free growing meristems and most of those seen were devoid of starch and had no organized lamellar structure. The osmiophillic (lipid) bodies of the type seen in the dormant and SD apical meristems were small and scarce in the FGLD meristems. While the nuclei in the central mother-cell zone were large and euchromatic, those in the peripheral zone and rib- pith meristem were highly heterochromatic. These nuclei appeared to be smaller and more elongate or flattened as well. The vacuoles in the pith-rib meristem and the peripheral zone cells were fewer and smaller than those of the central mother-cell zone. There were fewer starch grains seen in the plastids of the peripheral zone cells than in the pith-rib meristem cells (Figure 5.7D,F). Both the pith-rib meristem and the peripheral zone cells had denser cytoplasm than the central mother-cell zone cells, this appeared to be due to a higher ribosome density. A few sampled seedlings, all from NUR treatments, had set bud and had not reflushed under LD (NGLD). While the buds of these seedlings had the same external appearance as the SD buds, no preformed shoot was found within. Some contained a few partly elongated needles and some contained only bud-scales. In all cases, the last formed primordia appeared to be bud-scales (Figure SJ”. The meristems in these buds were larger than either those of the SD Figure 5.8 150 Light and transmission electron micrographs of the shoot apical region of a NUR seedling not resuming free growth under LD photoperiod. A) Light micrograph of a median longitudinal section of the apical meristem and last formed bud scale primordia (bs). Note lack of vascular differentiation in these primordia. There are relatively few tannin filled cells in the pith region . Bar = 250 um. B) Higher magnification light micrograph of same apical meristem. Note small vacuoles and high concentration of refractile bodies. Bar = 80 pm. C) Transmission electron micrograph of an apical- initial central mother-cell zone cell. Note small vacuoles (v), euchromatic nucleus (hn) with prominent nucleolus (n) and starch free plastids (p) The light area (cw) in the upper left corner is a thick region of the cell wall. Bar = 5 pm. In Central mother-cell zone cells showing slightly contorted cell walls, numerous plastids devoid of starch grains and numerous osmiophillic (lipid) bodies (lb). The vacuoles (v) present in these cells were small and sometimes had inclusions. Plasmodesmata (pd) were evident in the thinner areas of the cell walls. Mitochondria (m) were also abundant in these cells. Bar =5 um. E) Peripheral zone cells. These cells had heterochromatic nuclei Hun, small vacuoles, and no starch in the plastids. Bar = 5 pm. F) Rib-pith meristem cells. Cell wall distortion was most evident in the rib-pith meristem. Plastids in this region also had fairly large starch grains (8). Bar = 5 pm. 152 treatments or the FGLD seedlings (cf. Figures 5.5 and 5:”, at the end of the experiment. They appeared to be quite similar to those SD meristems sampled during bud-scale initiation. The zonation of NGLD meristems was not as distinct as that of the FGLD meristems. Their fine structure was roughly intermediate between the FGLD meristems and those of the SD seedlings. The cells of the central mother-cell zone, while having large euchromatic nuclei, had smaller vacuoles than those of the FGLD seedlings. The plastids had few starch grains, but there was an abundance of lipid bodies. These were sometimes aggregated on the margins of vacuoles (Figure SJ”. Nuclei of the cells of the peripheral zone and pith-rib meristem were heterochromatic as with the other treatments. The cell turgor seemed to be intermediate as well. The lower turgor was more evident in the pith-rib meristem cells and the peripheral zone cells, where some cell wall wrinkling was seen (Figure 5.8Efifl. DISCUSSION Initial observations The time to bud-break in this study was considerably shorter than that reported previously for spruce seedlings, whose Chilling requirement was fiilfilled, placed in conducive environments (3.3. Nienstaedt, 1966; Young and Hanover, 1977b). The anatomical differences between the last sample»(Dormant, unchilled) and initial sample which 153 had been chilled in storage indicate that the first sample buds were far from dormant. In mature Engelmann spruce, for example, mitotic activity in the apical meristem and elongation of the preformed shoot precedes visible bud break by more than a month (Harrison and Owens, 1983). This is also the case in Sitka and white spruce and many other determinate-growth northern conifers (Owens and Molder, 1973a,b, 1976; Owens, 3; 31., 1977; Owens, 1984). Mitotic activity is, perhaps, the best currently applicable indicator of growth resumption in tree meristems (Cecich, 1980). Physiological changes probably precede Observable mitotic activity in most conifer species. In the case of the cells in the pith of the preformed shoot of Scots pine (Pinus sylvestris Lu)'buds, for example, there are changes in the carbohydrate content or composition detectable about seven days prior to resumption of mitotic activity (Hejnowicz, 1979). Starch accumulation and/or redistribution is also seen throughout seedlings of Scots pine prior to bud-break (Ericsson, 1984). Histochemical analyses, such as those performed by Vanden Born (1963) and Fosket and Miksche (1966) also indicate that considerable physiological changes occur without mitotic activity in apical meristems. Although the seedlings in this study, as received, all had completely closed buds, considerable post dormancy activity had been underway within them. This advanced L54 development, in conjunction with the warmer than natural temperatures in the growth room, probably precipitated the rapid bud break. While the more advanced development of the of the AG seedlings might have resulted from several interacting cultural or storage (Arronsson, 1975; Lavender, 1980) conditions, it may have resulted partly from their age. One-year-old nursery grown spruce seedlings undergo more post differentiation needle development in the fall than do older seedlings (Burley, l966a,b; Chapter 3d. They also tend to break bud sooner under conducive conditions (Nienstaedt, 1966; Young and Hanover, 1977b). The higher number of starch filled plastids in the apparently less advanced meristems seems unusual. In Scots pine buds, starch usually decreases in the fall, is lowest in the winter, then is detectable again in the spring (Hejnowicz, 1979). A similar trend is seen in the needles (Martin and Oquist, 1979). I have noticed a similar trend during the annual cycle in mature blue spruce, growing in lower Michigan (Heckman, unpublished data). Small starch grains were abundant in the central mother cell zone and developing pith in the elongating shoots prior to bud break. By mid November, very few were present. The latter observations are consistent with the theory developed early in this century by Lidforss (1896, 1907) and briefly discussed by Steponkus (1984) that starch is hydrolized during dormancy induction to provide sugars which have a 155 cryoprotectant role. The former observation, that of this investigation, may have resulted from the disruption of the natural bud phenology, especially with respect to the temperature regime, caused by nursery storage. In the initial sample, the less advanced meristems also appeared to be less hydrated than those more advanced towards bud-break. Evidence of this is given by the wrinkled appearance of the cell walls, denser cytoplasm, and small vacuoles (Figure SJD. This may have been a residual characteristic of the hardened, post-dormant state. Lower intracellular cellular water content is a commonly observed attribute of chilling-resistant dormant plant material (Weiser, 1970; Levitt, 1980). Since the dehydrated appearance was not seen in all initially sampled meristems, and all meristems at each sample time were processed identically, it does not appear to be a preparation artifact. Chemically-caused dehydration damage in plant microtechnique is usually characterized by the separation of the plasma membrane from the cell wall and this was not seen. Further, the ultrastructural differences between the advanced and less advanced buds, associated a wide range of biochemical and physiological processes would be hard to induce quickly and simultaneously as artifacts. Cecich, (1977, Figures 1 and 2) shows the shoot apical meristem of Pinus banksiana seedlings prepared for light microscopy by CRAF fixation and paraffin techniques and prepared for electron microscopy essentially as described 156 above. In the TEM preparation, the cells have a similar appearance, where as the paraffin preparation shows no indication of wrinkled cell walls. This effect of paraffin technique may explain why a shrunken appearance in dormant spruce meristem cells has not been reported previously, in Spite of many excellent phenological studies Q53; Owens and Molder, 1976; Owens, 33 31., 1977; Harrison and Owens, 1983). The heterochromatic content of the nuclei and nuclear size also varied with the apparent phenological status of the initially sampled meristems. Heterochromatin has been reported to be relatively inactive in RNA synthesis (Berlowitz, 1965). This is consistent with the dormant condition. The heterochromatin seen in the nuclei in the dormant meristems may be constituative, however, and perhaps due to smaller nuclear volumes seen in these meristems (Owens and Molder, 1973; Riding and Gifford, 1973; Cecich, 1977). Bud burst and shoot elongation The phenology of the buds grown under SD was similar to that seen in spruce, and many other conifers with similar shoot phenology, in the natural environment. In the natural growth cycle of these trees, bud-scales are formed during budburst and shoot elongation in mature spruce, (Owens amd Molder, 1976; Owens,g£ 31., 1977; Harrison and Owens, 1983), Douglasfir (Pseudotsuga menzeseii) (Sterling, 1946; Owens amd Molder, 1973a), 157 western hemlock (Tsuga heterophylla (Raf.) SargJ (Owens and Molder, 1973b), and the true firs ( Abies gpd (Parke, 1959; Owens and Singh, 1982; Owens, 1984). Throughout the ranges of these species, this occurs under the longest photoperiods of the year. In the SD material in this experiment, grown under 12 hr photoperiod, most seedlings had visible bud scales by the second sample and all did by the third, taken at the end of shoot elongation. These were also the sample times during the experiment when the shoot apical meristem hadreached or was approaching its maximum volume under SD (Figure 5d“. Thus, bud initiation was not altered by the short photoperiods. The pattern of apical development under LD treatment was completely different. It also differed from that reported in germinant seedlings grown under LD (Gregory and Romberger, 1972a; Cannell, 1978; Chapter 2). In these studies, and other studies of the initial development of the conifer meristem (3.3. Tepper, 1964; Fosket and Miksche, 1966; Riding, 1972), the meristem gets bigger over time. In this study there was an overall decline. This may be attributed to both endogenous and environmental factors. The most probable environmental factor was the low PPFD. Under the LD treatment, the mean PPFD at the soil level was 35 pMm‘zsec'l or about 225 foot-candles. This is less than 10% of the saturation photon-flux-density for Engelmann spruce seedlings (Ronco, 1970; Tinus and McDonald, 158 1979). Blue spruce seedlings are reported to have similar or even higher saturation point (Tinus, 1979). While the PPFD was greater at mid-canopy of the seedlings, it was never greater than 100 ,uMm‘“2 sec‘l. This corresponds to considerably less total energy input (photon flux) than previously used for such experiments, even considering the 24 hr photoperiod. Shoot elongation in conifers is a major sink for the stored and new photosynthesis of the previous years shoot (Dickmann and Kozlowski, 1970; Loach and Little, 1973; Little, 1974; Chapter 4). The expansion of free growing stem units also represents a likely metabolic sink. Under the low light intensities used here these demands may not have been met. If the carbohydrate requirement for free grown stem unit elongation and that needed for meristem "reinvestment" Egggg Gregory and Romberger (1972a) were greater than that formed 92.22X2I the development of either or both might suffer. Needle differentiation without tissue replacement might reduce the size of the undifferentiated meristem in a manner analogous to that seen during late needle formation in mature spruce trees and seedlings (3.3. Owens, 1976; Owens st 31., 1977; Cannell and Cahalan, 1979; Harrison and Owens, 1983). Since there is less post initiation development in primordia formed under SD and almost no stem elongation (1.3. within the forming bud), one would expect a lower photosynthate demand. The lower than natural total quantum 159 flux might then have less of an effect the development of these buds. Final observations By the end of shoot elongation, in mature spruce trees, the apical meristem is covered by a protective bud. At this time in the annual cycle, needle primordia are initiated but do not elongate. This development of needle primordia continues until late autumn, a period of up to 4 months, after which the apical meristem is at its dormant size, mitotic activity ceases, and it shows the least zonation (Owens and Molder, 1973a, 1976; Owens, 33 31., 1977; Harrison and Owens, 1983). In germinant seedlings of white spruce, grown under AG conditions for 3 months, Pollard (l974a) found that most provenances had completed needle primordia initiation by 12 weeks when the seedlings were placed under short days with optimum temperatures and nutrients. In that study, Pollard also showed that most of the needle primordia formation occurred in the first six weeks of bud development. In this experiment, buds formed under SD treatments had begun to form scales by the sixth week, allowing somewhat less than 10 weeks for needle primordia initiation prior to the end of the experiment. The preformed shoots within the buds had formed well developed crowns by the last sample. In the determinate conifers that have this structure (see Lewis and Dowding, 1924; Venn, 1965), this occurs during late needle initiation (Parke,l959; Owens and Molder, 1973b, 160 1976; Owens, e_t.a_l., 1977; Pillia and Chacko, 1978; Harrison and Owens, 1983). This implies that the buds in this study had nearly all their preformed needle primordia differentiated. The differences in needle primordia number between the NUR and AG buds were probably due to the transplanting effects (Chapter 4). The apical meristems of the SD buds at the end of the experiment were similar to that of the less advanced buds at the beginning. The qualitative differences between the SD and FGLD seedling meristems were similar to those between the meristems of the more and less advanced buds at the initial sample. Apparently photoperiod can alter the carbohydrate, lipid and water status of the meristem itself in addition to (or conjunction with) controlling the fate and development of the primordia initiated. These characteristics, combined with the mentioned changes in the cytoplasmic density and nuclear staining seem to be responsible for the loss of distinct cytohistological zonation reported in dormant conifer meristems (Owens and Molder, 1973a; Cecich, 1977). The shrinkage of the vacuoles and wrinkled appearance of the cell walls seem to indicate that the meristem is at a lower state of hydration. The chemical binding or export of free water is often associated with the development of frost hardiness (Weiser, 1970; Levitt, 1980). It was not determined if these apparent changes of hydration were present throughout the seedling or just within the bud, 161 although cells of the bud-receptacle region (when not trimmed away) did not appear similarly shrunken. If the latter were true, it would further support the idea of the crown structure as a physiological barrier (Lewis and Dowding, 1924; Chalupa and Durzan, 1973; Jansson gt 31., 1983). If the former were true, a developmental photoperiodic effect on either root uptake and transport or a similar hardening mechanism through out the plant would be implied. The higher number of starch filled plastids in the cells of the SD meristems is incongruous with that seen in the apical meristems and preformed shoots of mature blue spruce in the late fall. As mentioned with respect to the initial sample, starch grains are more prevalent in the spring and disappear in the fall. This is thought to be related to the increase of free sugars (by hydrolysis) associated with cryoprotection (Weiser 1970; Arronsson gt a_l_., 1976; Levitt, 1980). In tobacco (Nicotiana tabacum) leaves. Far red light lowers the size and number of starch grains seen and increases the amount of soluble sugar. Red light has the opposite effect, implying a phytochrome mediated response (Kasperbauer and Hamilton, 1984). In the material in this investigation starch appeared to accumulate under SD conditions. Since phytochrome reverts to its red absorptive form (1.3. a similar effect to the far-red treatment described above) in the dark (Song, 162 1984), the SD response seen in this investigation suggests that the seasonal hydrolysis of starch to sugars in spruce might be triggered by temperature. This may in turn be a factor in the interdependence of photoperiod and temperature on the development of spruce seedling winter-hardiness (Arronsson, 1975; Christersson, 1978; Ierust and Cameron, 1982). In the lower elevation Himalayas, where the climate is seasonal but mild, Pillia and Chacko (1978) found that starch increased in October and decreased in January and February in Indian spruce (Picea smithiana (Wall.) Biossd buds. Other than this aspect, the rest of the bud phenology they described was similar to those reported for North American spruce species (loc. citJ. Indian spruce also appears to be less cold hardy than blue spruce (Rehder, 1940), further suggesting a relationship between the starch —sugar balance and the development of cold hardiness. At the end of the experiment, all LD AG meristems were free growing (FGLD). These meristems as described above were smaller than those of the initial sampleu This was also the case in most of the FGLD NUR meristems. It would appear that for free growth to have continued in these seedlings, either the rate of primordia formation would have needed to decrease or the volume of undifferentiated tissue in the meristems would have needed to increase. The NGLD meristems (Figure 5.8)1were anatomically' similar to the SD meristems, sampled during bud-scale 163 initiation. By the end of the experiment, however, the buds were similar in external appearance to those formed under short days. Removal of the bud scales revealed no preformed shoot formation. Either the meristem had stopped differentiating primordia after the normal number of bud scales had been formed and they had continued to grow or the primordia differentiated remained bud scales. Although (unfortunately) no counts were made of the individual bud scales of either LD or SD buds, it appeared as though the latter was the case. The bud cavities were filled with scales. If the continued production of bud scales in the NGLD seedlings did occur and it was not an effect of transplant shock, it may represent the mature response of the spruce shoot apical meristem to photoperiod. Under natural conditions, mature spruce apical meristems produce bud scales during the days with the longest photoperiod (loc. citJ. Since under short days, the first neoformed primordia always differentiated into bud scales, the mature photoperiodic response may involveethe inhibition of needle primordia initiation, rather than the induction of bud scales. In conclusion, shoot height, with in the range of the material used in this investigation, has no main effect on apical meristem phenology. Shoot apical meristem phenology appears to change as the seedlings age, although transplant shock may have affected this both quantitatively and 164 qualitatively. Rather than the loss of photoperiodic response, as seedlings mature, there appears to be a change in the photoperiodic effect on organogenesis at the apical meristem. Under short photoperiods, preformed shoot development appears to proceed to completion even in the absence of limiting temperatures but some histological changes, associated with chilling hardiness, do not seem to occur, under warm growth-room conditions. CONCLUSIONS AND RECOMMENDATIONS The results of this study indicate that spruce germinant seedlings, of a diverse genetic origin, appear to grow at nearly the same relative growth rate under propitious conditions. The large interspecific differences in dry weight appear to be due to initial seed size and germination vigor even after 6 months of culture. For at least the first 6 months, the growth of a relatively unrestricted spruce seedling is nearly exponential, its biomass growth-rate, at any time, being proportionate to its weight. That this rate cannot continue is also obvious. Its reduction is foreshadowed by the apparently asymptotic increase in height growth rate. In order for growth to proceed at an exponential rate, either all of the plant's mass must increase at the same unit rate 3.3. remain totally meristematic, or the unit rates of the meristematic areas must continue to increase. While neither of these possibilities makes biological sense, the observed rates do present some interesting implications for both containerized seedling production and an understanding of spruce seedling development. From the standpoint of greenhouse culture, the concept of even an initial exponential-growth rate, has some strong implications. The "lag" phase commonly observed during early development is apparently due to the low RER of about 165 166 extremely important at this point to avoid any circumstances that even slightly reduce vigor, as the reduction might be proportionally expressed throughout the rotation period. The results also imply that regardless of the species grown, efforts to secure the fastest germinating, highest weight seed should be made, for the fastest absolute growth. From the standpoint of further research, the most immediately promising areas would seem to lay at either end to the sample time reported here. Little information is available on the initial dynamics of germination in spruce, or what affects its initial growth rate. The very early growth-rate dynamics are often different than the average rates over periods such as this investigation (Hunt, 1982). Such information would perhaps be useful to those involved with small propagule sizes in vitro. At the other extreme, the existence of a nearly exponential growth rate after six months of growth, in light of the stabilizing height growth-rate and changes in tissue distribution in the apical meristem, also warrants further investigation. While the seedlings in this experiment never completely filled their containers with roots, the effects of root and shoot environment on long term experiments requires careful consideration in experimental design. A root medium that could be replaced periodically or continuously, such as a hydroponic system, might be advantageous in this respect, as would higher light intensities. 167 The shoot growth rate, on the other hand, approaches a nearly constant value, after about six months of accelerated growth. Plastochron index and height were very closely related in the time span of this experiment indicating a relatively uniform internode elongation, within each species. Since differences in height-growth rate between species, in this study, appeared to be more related to internode elongation than needle number, plastochron duration in spruce may be nearly the same under accelerated growth conditions. Growth analyses techniques of this type may prove useful in developing greenhouse or nursery crop monitering programs, in addition to basic information on how seedlings grow. Past work in spruce has indicated that the rate of needle initiation is strongly tied to the volume (or basal area) of the apical meristem in Norway spruce (Gregory and Romberger, l972a,b) and Sitka spruce (Cannell, 1978a) in these studies, the apical volume increased asymptotically over time. The anatomical results here indicate that a similar response is seen in other spruces. Later work by Gregory and Romberger (1977) and showed that vascular differentiation, as indicated by protoxylem initiation, occurred with chronometric uniformity rather than morphometric. This implies a possibly different "control system" for internode elongation than primordia initiation at the apex. The results from Chapter 4, that photoperiod did not affect internode elongation during flushing, in the 168 blue spruce seedlings tested, lends further credence to this hypothesis. It is interesting to note that the general effect of shortened growth cycles appeared to be retard seedling development rather than accelerate it. After more time under shortened growth cycles than it takes for two growth seasons of nursery culture, the apical development was no greater than a one-year-old seedling. The results of the flowering studies using similar shortened growth cycles by Dormling 33 31, (1968) have not, to my knowledge, been reported. From the standpoint of accelerating maturity in spruce, it would appear that systems allowing the most uninterrupted growth, hold more promise (3.3. Young and Hanover, 1976). Even in very short growth cycles, progressively more time is seems to be needed for bud formation. This may also be the case for free grown seedlings of progressively increasing size. In white spruce Pollard (1974b) found that taller seedlings within an age class had more bud needles after 10 weeks of short days, but that the relative advantage declined with age. If the older, taller seedlings took longer to complete bud set, they may have had less time left for needle initiation. Thus, the greenhouse practitioner may need to consider the coordination of three stem growth components, initial free growth height, the time to complete bud scale formation, and the time to initiate needle primordia. 169 Within 75 to 300 mm tall two-year-old (2+0) blue spruce, seedling height does not appear to limit free growth. Seedlings in the middle of this range were the only ones to show, quantitatively, free growth. However, the overall mean was significantly greater than the non- inductive control as well. Since all of the AG seedlings in this height range free grew under long days, height, alone, within this range, does not seem to affect free growth. Comparison of the shoot growth development in the nursery-grown seedlings to that of the AG seedlings illustrated some equivocality in the use of transplants in such studies. ‘While the shoots of the NUR seedlings were larger in most measured characteristics, and had more shoot growth potential their out-growth performance was relatively less than the AG "plugs" when grown indoors under either continuous or short day photoperiods. Ideally, this aspect of the change in photoperiod response should be clarified by using various aged seedlings, grown from seed in large pots. That growing transplants in the test pot for the year prior to the experiment (3.3. Nienstaedt,l966) may not be sufficient to remove transplanting effects is suggested by the field work of Burdett and others (1984). In this experiment, transplanted (2+1) seedlings appeared to develop fewer primordia than AG seedlings under short day. Anatomically, there were many differences between the apical regions of seedlings grown under long and short days. The short day response was the most varied less with the 170 size or age of the seedling than the long day treatments. Under short days, bud development followed the same general phenology as seen in mature spruce. Use of TEM preparation techniques yielded considerable new information at light and TEM levels. While these results should be replicated with material grown under more "normal" lighting, it appears that photoperiod, either directly or indirectly, alters the water and carbohydrate status of the bud as it forms. Starch accumulation occurred in the dormant SD buds. This is opposite of what is usually seen in northern temperate zone conifers under natural conditions. In light of the implications in seedling hardiness and growth potential chemical analyses should be undertaken along with further anatomical observations. Under long days, the apical meristem took on two different types of morphology related to the presence or absence of free growth. While the anatomy of the free growing meristems was qualitatively similar to the paraffin image, the TEM preparation technique, in spruce, suggests that the light staining seen in the CMZ of active meristems is not a lipid extraction artifact (sf. Cecich, 1980). Those seedlings not free growing under long days appeared to be in a state of prolonged bud set. There may be a change in photoperiodic response as the seedling ages, rather than a loss. Long days in mature spruce may inhibit needle primordia initiation. BIBLIOGRAPHY BIBLIOGRAPHY Arnott, J. T. 1979. 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