HORTSCIENCE 36(5):971–975. 2001.
Chlorophyll Fluorescence and Vegetative Propagation of Taxus Sarah E. Bruce1, D. Bradley Rowe2, and James A. Flore3 Department of Horticulture, Michigan State University, East Lansing, MI 48824-1325 Additional index words. yew, stem cuttings, rooting, stress, photochemical efficiency, Fv/Fm, Taxus ×media Abstract. Chlorophyll fluorescence over the course of stem cutting propagation was examined in 10 cultivars of Taxus ×media (Taxus baccata L. x T. cuspidata Sieb. & Zucc.), including ‘Brownii’, ‘Dark Green Pyramidalis’, ‘Dark Green Spreader’, ‘Densiformis’, ‘Densiformis Gem’, ‘Hicksii’, ‘L.C. Bobbink’, ‘Runyan’, ‘Tauntoni’, and ‘Wardii’. The fluorescence value measured was the ratio of variable over maximum chlorophyll fluorescence (Fv/Fm). This value reflects the maximum dark-adapted photochemical efficiency of photosystem II (PSII) reaction centers involved in photosynthesis and is an indirect measure of plant stress. The objective of this study was to examine Fv/Fm as a method for stock plant selection and for monitoring rooting progress of various cultivars. Fv/Fm varied significantly (P ≤ 0.05) among cultivars, initially and over time. However, there was significant overlap among some cultivars. The Fv/Fm decreased dramatically during cold storage, but usually returned to original levels after several weeks in the propagation beds. This appeared to be a reflection of the reduction of water stress as the cuttings formed roots. Initial stock plant Fv/Fm was not correlated (P ≤ 0.05) with rooting percentage, root number, root dry weight, or root length, indicating that Fv/Fm is not a reliable indicator of stock plant rooting potential. Visual assessment is just as reliable. The nursery industry supplies yew (Taxus sp. L.) for both ornamental and pharmaceutical uses. Most of these plants are propagated by rooting stem cuttings, and success is often related to stock plant health (Moe and Andersen, 1988). Many stresses that affect plant health, such as water stress, pollution (Saarinen, 1993; Saarinen and Liski, 1993), photoinhibition (Hallgren et al., 1990; Lichtenthaler and Rinderle, 1988; Welander et al., 1994), freeze damage (Fisker et al., 1995), and mineral nutrient and carbohydrate status (Rowe et al., 1999), also influence potential adventitious rooting of cuttings severed from these plants. Currently, stock plants are evaluated from visual observations, but if a quick, reliable quantitative method of determining potential rooting of cuttings based on the condition of a specific stock plant was available for propagators, then rooting success could be predicted prior to an investment in time, labor, and resources. Chlorophyll fluorescence measurements may be a method for evaluating stock plants. Chlorophyll fluorescence occurs when excess Received for publication 23 Aug. 2000. Accepted for publication 5 Mar. 2001. This paper is a portion of a MS thesis submitted by S.E. Bruce. This research was funded by: Zelenka Nursery in Grand Haven, Mich., International Plant Propagator’s Society, Michigan Nursery and Landscape Association, and Michigan Agricultural Experiment Station. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must be hereby marked advertisement solely to indicate this fact. 1 Graduate Research Assistant. 2 Assistant Professor; to whom reprint requests should be addressed. E-mail address:
[email protected] 3 Professor.
light energy, absorbed by chlorophyll a, is dissipated by being re-emitted as fluorescence. Since chlorophyll fluorescence levels are tied to the maximum dark-adapted photochemical efficiency of photosystem II (PSII), they often can serve as a general measurement of plant photosynthetic potential (Krause and Weis, 1991; Lichtenthaler, 1988). In fact, the Fv/Fm ratio has become an important and easily measurable parameter of the physiological state of the photosynthetic apparatus of intact plant leaves (Krause and Weis, 1991). The emitted light signal follows a general intensity pattern known as the Kautsky Effect. Pre-darkened samples, with a minimum fluorescence level (Fo), exhibit a rapid increase in fluorescence to a maximum value (Fm) upon exposure to a light source. A common parameter used in stress studies is Fv/Fm, Fv being the variable fluorescence, calculated by subtracting Fo from Fm (Krause and Weis, 1991). Numerous studies have shown Fv/Fm, and other chlorophyll fluorescence parameters, to be effective measures of the photoefficiency of PSII. Björkman and Demmig (1987) found a positive linear correlation between Fv/Fm and the optimum quantum yield of PSII in a variety of stressed plants. Although Fv/Fm provides a rapid and nondestructive measurement of plant stress, few data are available on its usefulness in propagation, in particular as an indication of stock plant rootability. Van Huylenbroech and Debergh (1992) used chlorophyll fluorescence (Fv/Fm) to examine stress levels during the acclimation of Transvaal daisy (Gerbera jamesonii Bol. ex Adlam) following micropropagation. They observed two periods of stress when fluorescence values decreased: first when the plants were not yet rooted and a
second period when plants were transferred from weaning conditions (high relative humidity) to normal greenhouse conditions. In addition, Sallanon et al. (1998) found that high light intensity and water stress decreased Fv/ Fm during acclimation of micropropagated Rosa ×hybrida. Similarly, Genoud et al. (1999) rooted micropropagated shoots of Rosa ×hybrida on solid sucrosed media (MS) under an irradiance (PPFD) of 45 µmol·m–2·s–1 or on a liquid hydroponic solution (MH) at 100 µmol·m–2·s–1. During the rooting phase and after 21 d of acclimation, Fv/Fm was higher for plantlets cultivated under MH conditions, but no differences remained after 42 d. In a study with conventional rooted cuttings of the woody species Cordia alliodora (Ruiz & Pav.) Oken, Mesen et al. (1997) reported that Fv/Fm decreased to its lowest value 2 d after insertion of cuttings, but increased by day 5 and remained high for the remainder of the experiment. They also found strong positive correlations between final rooting percentage (week 6) and mean Fv/Fm at weeks 1, 2, and 3. To our knowledge, no studies have attempted to correlate initial stock plant fluorescence with subsequent rooting of stem cuttings. Therefore, the objectives of this study were to examine the differences in chlorophyll fluorescence among 10 cultivars of Taxus ×media, and changes in chlorophyll fluorescence over the course of their propagation, and to correlate initial chlorophyll fluorescence values with rooting. Materials and Methods Ten cultivars of Taxus ×media were selected for this 2-year study: ‘Brownii’, ‘Dark Green Pyramidalis’ (first year only), ‘Dark Green Spreader’, ‘Densiformis’, ‘Densiformis Gem’, ‘Hicksii’, ‘L.C. Bobbink’, ‘Runyan’, ‘Tauntoni’, and ‘Wardii’. Trials were performed at two locations, East Lansing, Mich. (Michigan State Univ.), and Grand Haven, Mich. (Zelenka Nursery), for the first year of the study, and at Grand Haven the second year. Propagation procedures were similar in all trials. In early fall, 15–20 cm cuttings were severed from field-grown plants at Grand Haven. Cuttings were bagged in plastic and placed in cold storage in the dark at either 2.5 or 5 °C. Initial chlorophyll fluorescence measurements were recorded prior to placement of cuttings in coolers. After 4 weeks in cold storage, cuttings were cut to 11.4 cm (apical and basal portions removed). Bases were then treated with a mixture of indole-3-butyric acid (IBA) at 1700 mg·L–1 and naphthaleneacetic acid (NAA) at 1100 mg·L–1 dissolved in water (Woods Rooting Hormone; Earth Science Products Corp., Wilsonville, Ore.) and inserted 3 cm deep into a 100% perlite rooting substrate. Cuttings were stuck in greenhouse benches or trays in rows 3.8 cm apart with 1.3 cm between individual cuttings within each row. In spring, cuttings were gently uprooted, total root length per cutting was rated [0 = less than 2.5 cm (1 inch); 1 = less than 5.1 cm (2 inch); 2 = less than 7.6 cm (3 inch); 3 = greater
than 7.6 cm], and root numbers were recorded. Roots were dried at 60 °C for 5 d and weighed. Chlorophyll fluorescence measurements were recorded periodically throughout the propagation process. Some cultural and greenhouse conditions differed between the two locations. At Grand Haven, immediately after sticking, cuttings were drenched with pentachloronitrobenzene (PCNB; Lesco, Rocky River, Ohio) mixed at 0.6 g·L–1 and applied at 3.05 L·m–2. For the remainder of the time that cuttings were held in the rooting beds, a rotation of fungicides was applied every 21 d: thiophanatemethyl [dimethyl 4,4´-o-phenylenebis(3thioallophanate)] (Cleary’s 3336; Cleary Chemical Corp., Dayton, N.J.) mixed at 0.8 g·L–1 and applied at 3.05 L·m–2; 5-ethoxy-3(trichloromethyl)-1,2,4-thiadiazole (Terrazole‚ 35% WP; Uniroyal Chemical Co., Middlebury, Conn.) mixed at 0.6 g·L–1 and applied at 3.05 L·m–2; and 4-(2,2-difluoro-1,3benzodioxol-4-yl-)-1H-pyrrole-3-carbonitrile (Medallion; Novartis Crop Protection, Greensboro, N.C.) mixed at 0.2 g·L–1 and applied at 3.05 L·m–2. Cuttings were fertilized with water-soluble 12N–18.3P–8.3K (Greencare 12–42–10; Greencare Fertilizer, Chicago) mixed at 3.0 g·L–1 and applied at 3.05 L·m–2, beginning at the end of February once cuttings had formed roots. Fertilization was continued every 21 d in conjunction with fungicide treatments until cuttings were harvested. Cuttings at East Lansing received no fungicide treatments or fertilizer. Grand Haven greenhouses were covered with Cloud Nine polyethylene film (Huntsman Packaging Corp., Newport News, Va.), and East Lansing houses were glass. Experiments at both locations were conducted under natural irradiance and daylength. Grand Haven propagation benches were 15 cm deep with bottom heat (21 ± 2 °C). East Lansing growth trays were 7.6 cm deep and received no bottom heat. Greenhouse air temperatures at Grand Haven were maintained at a mean temperature of 13 °C with a minimum of 10 °C and a maximum of 18 °C. By the time greenhouse temperatures reached 18 °C during April, the rooting phase was completed. East Lansing air temperatures were more variable, often exceeding 21 °C and sometimes reaching 32 °C during sunny days. High relative humidity was maintained in the greenhouses by wetting the concrete walkways two to five times per day, depending on weather conditions. There was no intermittent mist at either location. The 1997–98 trial at East Lansing used a randomized complete-block design with six blocks, 10 cultivars, and 10 cuttings within each block/cultivar treatment, for a total of 600 cuttings. Cuttings were collected 14 Oct. and kept in cold storage at East Lansing (5 °C) until stuck on 22 Nov. Harvest took place 21 May. Chlorophyll fluorescence was measured on 10 cuttings per cultivar, at severance and twice during cold storage (30 Oct. and 18 Nov). During rooting, four readings were recorded per cultivar, within each block on 3 and 18 Dec., 6 and 29 Jan., 3 Mar., 6 Apr., and 6 May.
Fig. 1. Chlorophyll fluorescence in 10 Taxus cultivars over the course of propagation at (A) Grand Haven, Mich. (Zelenka Nursery), and (B) East Lansing, Mich. (Michigan State Univ.), during 1997–98. (A) Cuttings were collected from stock plants in the field at day 0, placed in cold storage at 2.5 °C, stuck at day 37 in rooting beds (100% perlite) with bottom heat at 21 ± 2 °C, and harvested at day 206. Greenhouse temperatures were maintained at 13 °C with a minimum of 10 °C and a maximum of 18 °C. (B) Cuttings were collected at day 0, placed in cold storage at 5 °C, stuck at day 35 in rooting beds (100% perlite) with no bottom heat, and harvested at day 205. Greenhouse temperatures often exceeded 21 °C. For both locations, chlorophyll fluorescence (Fv/Fm) was measured periodically until harvest. The first three points per cultivar are means of 10 measurements, while all other points represent means of 24 measurements. Vertical bars represent SE for entire population within each sampling date.
The 1997–98 trial at Grand Haven was designed similarly to the East Lansing trial; however, only four of the six blocks were harvested. Cuttings were collected 14 Oct., stored at Grand Haven in open buckets at 2.5 °C, and stuck 20 Nov. Harvest took place 27 May. Chlorophyll fluorescence was measured on 10 cuttings per cultivar, at severance and twice during cold storage (30 Oct. and 20 Nov.). Four readings were recorded per cultivar, within each block, on 4 Dec., 8 and 30
Jan., 27 Feb., 3 Apr., and 7 May. The 1998–99 trial at Grand Haven was performed similarly to the 1997–98 Grand Haven trial with a few exceptions. Cuttings were severed 29 Oct. and a fluorescence reading recorded for each individual cutting. Cuttings were rolled in plastic and placed in cold storage (5 °C) at East Lansing until Dec. 1, when they were stuck at Grand Haven. The experimental design was a randomized complete block with four blocks, nine cultivars,
and 15 cuttings per block/cultivar treatment for a total of 540 cuttings. The selection ‘Dark Green Pyramidalis’ was not tested because of a shortage of material. Chlorophyll fluorescence readings were recorded on each individual cutting on 30 Oct., 30 Nov., and 10 Mar., and a sample of six readings per cultivar, within each block, was taken 7 Jan. and 3 Feb. Needles for all tests were randomly selected. Cuttings were harvested 10 Mar., 3 months earlier than the previous year, in order to maximize differences in rooting percentage among cultivars. Chlorophyll fluorescence measurements were recorded with a Plant Efficiency Analyzer (PEA) fluorometer (Hansatech Instruments Ltd., Norfolk, England, U.K.). For each measurement (Fv/Fm), a randomly selected needle was dark-adapted for 15 min using the manufacturer’s plastic/foam clips. Chlorophyll fluorescence was usually measured the same day as the needles were collected, although occasionally logistics required detached needles to be stored overnight at 5 °C in a germination tray covered with moist paper towels. Needles were acclimated to room temperature at least 1 h prior to fluorescence measurements. No distortion of values was observed as a result of overnight storage. Fluorescence illumination was provided by an array of six high-intensity light-emitting diodes (LED), which were focused onto the sample surface to provide even illumination over the exposed leaf surface. Red actinic light of a peak wavelength of 650 nm was provided and readily absorbed by the chloroplasts. A fluorometer actinic light level of 1200 µmol·m–2·s–1 was determined sufficient to saturate PSII, according to our preliminary tests per the manufacturer’s instructions (Hansatech Instruments, 1997). An algorithm was used to determine the line of best fit through the initial 8–24 data points at the onset of illumination. This line of best fit was then extrapolated from time zero to determine F0 (initial or minimal fluorescence), and Fm (maximum fluorescence) was obtained at the same light intensity when the primary electron acceptor from PSII (QA) became fully reduced. Variable fluorescence (Fv) was calculated by subtracting F0 from Fm, and Fv/Fm was calculated from the Plant Efficiency Analyzer (PEA) fluorometer (Hansatech Instruments, 1997). Statistical analysis. Data for rooting and chlorophyll fluorescence were subjected to ANOVA, and Waller–Duncan Bayesian kratio multiple comparison t tests were performed on each trial of the harvest. Simple correlation coefficients were calculated for individual cutting chlorophyll fluorescence and harvest data for the 1998–99 season (proc anova, proc corr, SAS/PC software; SAS Institute, Cary, N.C.). Results and Discussion Initial chlorophyll fluorescence. Chlorophyll fluorescence varied significantly (P ≤ 0.05) among cultivars during both seasons; however, ranges for individual cultivars over-
Fig. 2. Chlorophyll fluorescence in nine Taxus cultivars over the course of propagation at Grand Haven, Mich. (Zelenka Nursery, 1998–99). Cuttings were collected from stock plants in the field at day 0, placed in cold storage at 2.5 °C, stuck at day 32 in rooting beds (100% perlite) with bottom heat (21 ± 2 °C), and harvested at day 132. Greenhouse temperatures were maintained at 13 °C with a minimum of 10 °C and a maximum of 18 °C. Chlorophyll fluorescence (Fv/Fm) was measured periodically until harvest. The first three points per cultivar are each a mean of 60 measurements, while all other points represent means of 24 measurements. Vertical bars represent SE for entire population within each sampling date.
Table 1. Rooting percentage and root dry weights of 10 cultivars of Taxus ×media at East Lansing (1997– 98), and Grand Haven, Mich. (1998–99). Each value is the mean of 60 observations. East Lansing Grand Haven Cultivar Rooting (%) Dry wt (mg) Rooting (%) Dry wt (mg) Brownii 47 ez 166 bc 97 a 84 a Dark Green Pyramidalis 76 bc 87 e ----Dark Green Spreader 62 d 117 de 49 e 45 bc Densiformis 97 a 202 ab 45 e 33 cd Densiformis Gem 85 ab 225 a 83 b–d 57 b Hicksii 65 cd 131 cd 87 a–c 75 a L.C. Bobbink 32 f 42 f 95 ab 87 a Runyan 70 cd 184 ab 47 e 53 b Tauntoni 57 de 133 cd 75 cd 23 d Wardii 88 ab 223 a 72 d 58 b z Mean separation within cultivars by Waller–Duncan Bayesian k-ratio multiple comparison t tests, P ≤ 0.05.
lapped extensively (Figs. 1 and 2). Initial values for 1997–98 ranged from 0.730 (relative units) for ‘Wardii’ to 0.873 for ‘Dark Green Pyramidalis’. With the exception of ‘Wardii’, subsequent readings within each cultivar for the 1997–98 year never exceeded these initial numbers at either location. Initial values for 1998–99 ranged from 0.778 for ‘Brownii’ to 0.833 for ‘Dark Green Spreader’. These readings are close to those reported for nonstressed, healthy conifers (Björkman and Demmig, 1987). Chlorophyll fluorescence over the course of propagation. Changes in Fv/Fm over the course of propagation were measured biweekly and then monthly in the first-year trials at Grand Haven (Fig. 1A) and East Lansing (Fig. 1B), and monthly in the second-year trial at Grand Haven (Fig. 2). Generally, Fv/Fm was highest at the time of severance, declined during cold storage and sticking, and then
increased as rooting occurred and growth resumed. During the 1997–98 study at Grand Haven, there was a clear decline in Fv/Fm over the 37 d of storage as the cuttings entered dormancy (Fig. 1A). This decline continued for more than 13 d after sticking, reflecting new stresses in the propagation process itself. The decrease in Fv/Fm during rooting are the result of limited water availability in the absence of roots, thus causing changes in water potential that affect stomatal opening and photosynthesis. Rooting occurred ≈35 d after sticking, at which point Fv/Fm increased. Time of rooting was verified by periodically pulling on random cuttings. Fv/Fm continued to increase until 1 month before harvest, when it dropped slightly. This drop could be caused by any number of factors, such as photoinhibition, heat stress, or water stress, associated with the warm, sunny May weather.
In the 1997–98 trial at East Lansing (Fig. 1B) and the 1998–99 trial at Grand Haven (Fig. 2), Fv/Fm again decreased during cold storage. These declines exhibited a wider range in Fv/Fm among cultivars than was observed in the 1997–98 Grand Haven data, probably because of warmer storage conditions (5 vs. 2.5 °C), suggesting differences in optimal storage temperatures among cultivars. In both these trials Fv/Fm increased quickly after sticking and peaked before rooting occurred ≈35 to 40 d after sticking. Fv/Fm then gradually declined until harvest. This pattern probably reflects warmer greenhouse conditions in these two trials; temperatures in the East Lansing greenhouse often exceeded 21 °C on sunny days and the unseasonably warm and sunny weather during the 1998–99 spring prevented keeping greenhouse temperatures at Grand Haven as cool as desired. Rooting. At East Lansing in 1997–98, rooting percentages ranged from 32% for ‘L.C. Bobbink’ to 97% for ‘Densiformis’, with much variation within cultivars (Table 1). Root dry weight ranged from 42 (‘L.C. Bobbink’) to 225 mg (‘Densiformis Gem’) per cutting (Table 1) and root numbers from 2.5 (‘L.C. Bobbink’) to 15 (‘Densiformis’) (data not presented). Differences in these measurements were evident among cultivars; however, individual cultivar means were generally indistinct. Root dry weight, number, and length provide information on the quality of rooted cuttings, which could influence future growth when planted in the field as liners. The three measurements were significantly correlated (R > 0.9; P ≤ 0.05), so only data for root dry weight are presented. In 1997–98, extremely high rooting percentages were recorded at Grand Haven. There were no detectable differences in rooting percentages among cultivars, which ranged from 97.5% to 100% (data not presented). Cultivars differed in dry weight and root length, both of which were higher than at East Lansing, and root numbers, which were twice as high as at East Lansing (data not presented). These results may be a response to the more favorable environmental conditions at Grand Haven, where cuttings were subjected to less environmental stress, as well as the longer propagation period. The greater root dry weight and length probably reflect the earlier rooting of cuttings and the greater depth of the propagation beds at Grand Haven. Furthermore, warmer temperatures in East Lansing forced a flush of shoot growth, potentially depleting stored carbohydrates that might otherwise have been allocated to root formation (Veierskov, 1988). The 1998–99 Grand Haven cuttings were harvested 132 d after cutting, instead of 225 d as in the previous year, in order to maximize differences among cultivars when compared to the 1997–98 Grand Haven trial. Rooting percentages ranged from 45% (‘Densiformis’) to 97% (‘Brownii’) (Table 1). Dry weights (Table 1), root numbers, and root lengths (data not presented) were lower during 1997–98, probably because of the earlier harvest date. Even though ‘Dark Green Spreader’ (49%), ‘Runyan’ (47%), and ‘Densiformis’ (45%)
rooted poorly, the results from the previous year suggest that with sufficient time, they would have rooted in high percentages. Unexpectedly, the relationships among cultivars differed greatly in the three trials. The poorest-rooting cultivars at East Lansing in 1997–98 were some of the best-rooting cultivars at Grand Haven in 1998–99. Environmental or environment/cultivar interactions played greater roles in determining chlorophyll fluorescence and harvest characteristics than did cultivar. One can only speculate on the many environmental factors that may have differed among the trials, years, and locations. Differing greenhouse conditions were surely a factor between the two locations, and the 2 years were accompanied by seasonal weather differences. Cultivar effects on time of root initiation may have come into play in the early harvest imposed the second year, and results may not be representative of what final May harvest results would have been. Correlations. For the 1998–99 data, each individual cutting was associated with a specific collection, sticking, and harvest Fv/Fm reading, and specific harvest data. No strong correlations were found, either experimentwide or within cultivars, between any of the Fv/Fm measurements and harvest data. However, some weak correlations existed at the P ≤ 0.05 level, but these correlations, although statistically significant, are too small to be commercially useful. Stock plant fluorescence and rooting would likely be correlated if a range of healthy to unhealthy stock plants were evaluated. In a preliminary study, some cuttings were chosen from plants exhibiting visible stress, but the resultant chlorophyll fluorescence readings were too low to measure. Chlorophyll fluorescence is an excellent early indicator of stress, but long-term effects of stress can be assessed visually. Short-term stress that is not yet visible would probably be measurable by Fv/Fm measurements and may in turn influence rooting, but even though there were some differences in initial Fv/Fm measurements in our study, all of our chosen stock plants were relatively healthy. Regardless, a visual observation of the stock plant is just as reliable and practical for predicting rooting. In addition, although differences in Fv/Fm existed among cultivars, they were not sufficiently distinct or consistent from experiment to experiment. Trends in Fv/Fm values over time were highly affected by local environmental conditions. This made comparisons between years and locations difficult because of the multitude of factors to consider. It also complicates attempts to use chlorophyll fluorescence as a stock plant quality measure, since each field would have its own unique set of local conditions. In conclusion, there was no strong correlation between chlorophyll fluorescence, measured as Fv/Fm, and rooting percentage, root number, root dry weight, or root length in the 10 Taxus cultivars examined. This may indicate that the quantum efficiency of PSII is not a significant factor in determining Taxus rooting characteristics. Inadequate control of varia-
tion is a potential source of error. Chlorophyll fluorescence measurements are affected by temperature differences, photoinhibition, and other seasonal environmental effects, as well as by top vs. bottom leaf surfaces, sun vs. shade leaves, needle age, storage time before measurement, and dehydration (Binder et al., 1997; Jiang et al., 1999; Krause and Weis, 1991; Lichtenthaler, 1988; Mohammed et al., 1995). Although the chlorophyll fluorescence parameter used, Fv/Fm, can be relatively consistent under some conditions (Björkman and Demig, 1987), it may not have been the best parameter for our purposes. Nevertheless, we do not consider chlorophyll fluorescence measurements to be a practical method for determining stock plant rooting ability in Taxus. Visual assessment is just as reliable as chlorophyll fluorescence measurements for selecting stock plants for cutting material. Literature Cited Binder, W.D., P. Fielder, G.H. Mohammed, and S.J. L’Hirondelle. 1997. Applications of chlorophyll fluorescence for stock plant quality assessment with different types of fluorometers. New Forests 13:63–89. Björkman, O. and B. Demmig. 1987. Photon yield of O2 evolution and chlorophyll fluorescence characteristics at 77K among vascular plants of diverse origins. Planta 170:489–504. Fisker, S., R. Rose, and D.L. Haase. 1995. Chlorophyll fluorescence as a measure of cold hardiness and freezing stress in 1+1 Douglas-Fir seedlings. Forest Sci. 41(3):564–575. Genoud, C., A. Coudret, C. Amalric, and H. Sallanon. 1999. Effects of micropropagation conditions of rose shootlets on chlorophyll fluorescence. Photosynthetica 36(1–2):243–251. Hallgren, J.E., T. Lundmark, and M. Strand. 1990. Photosynthesis of Scots pine in the field after night frosts during summer. Plant Physiol. Biochem. 28:437–445. Hansatech Instruments Ltd. 1997. Operating instructions for Plant Efficiency Analyzer (PEA) advanced fluorescence analysis. Hansatech Instruments Ltd., Norfolk, England, U.K. Jiang, H., G.S. Howell, and J.A. Flore. 1999. Efficacy of chlorophyll fluorescence as a viability test for freeze-stressed woody grape tissues. Can. J. Plant Sci. 79:401–409. Krause, G.H. and E. Weis. 1991. Chlorophyll fluorescence and photosynthesis: The basics. Annu. Rev. Plant Physiol. Plant Mol. Biol. 42:313–349. Lichtenthaler, H.K. 1988. Applications of chlorophyll fluorescence. Kluwer Academic, Dordrecht, The Netherlands. Lichtenthaler, H.K. and U. Rinderle. 1988. Chlorophyll fluorescence signatures as vitality indicator in forest decline research, p. 143–149. In: H.K. Lichtenthaler (ed.). Applications of chlorophyll fluorescence. Kluwer Academic, Dordrecht, The Netherlands. Mesen, F., A.C. Newton, and R.R.B. Leakey. 1997. The effects of propagation environment and foliar area on the rooting physiology of Cordia alliodora (Ruiz & Pavon) Oken cuttings. Trees 11:404–411. Moe, R. and A.S. Andersen. 1988. Stock plant environment and subsequent adventitious rooting, p. 214–234. In: T.D. Davis, B.E. Haissig, and N. Sankhla (eds.). Adventitious root formation in cuttings. Dioscorides Press, Portland, Ore. Mohammed, G.H., W.D. Binder, and S.L. Gillies. 1995. Chlorophyll fluorescence: A review of its
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