Morphological and physiological markers of juvenility and maturity in shoot cultures of oak (Quercus robur and Q. petraea)

Transcription

1 Tree Physiology 18, Heron Publishing----Victoria, Canada Morphological and physiological markers of juvenility and maturity in shoot cultures of oak (Quercus robur and Q. petraea) E. MCGOWRAN, 1 G. C. DOUGLAS 2 and M. PARKINSON 1,3 1 School of Biological Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland 2 Teagasc, Kinsealy Research Centre, Dublin 17, Ireland 3 Author to whom correspondence should be addressed Received August 30, 1996 Summary Evaluation of potential techniques for rejuvenating oak shoots requires robust quantitative markers for juvenile and mature plants. To identify suitable in vitro markers, shoot cultures of Quercus species of juvenile, adolescent and mature origin were screened for a range of morphological and physiological markers of juvenility and maturity. Criteria examined included angle of the shoot to the horizontal, stem length, stem diameter (tip, mid, base), leaf number, scale leaf number and shoot number. Image analysis was also carried out to determine leaf area, size, perimeter and breadth and length of leaves. Mature Q. robur L. clones had a larger mid-stem and tip diameter than juvenile clones, whereas mature Q. petraea ex Liebl. clones were characterized by plagiotropic growth and larger mid-stem and tip diameters compared with juvenile clones. Based on discriminant analysis of the data, we propose the following formulae for discrimination of juvenile and mature shoots, where a negative value for the discriminant score (D) indicates juvenility. For Q. petraea: D = SA TD MSD, (1) where SA is stem angle ( ), TD is tip diameter (mm) and MSD is mid-stem diameter (mm). For Q. robur: D = TD MSD. (2) Quercus robur clones derived from stump sprouts and designated as juvenile had a negative D value suggesting a juvenile status for these clones. Clones sourced from a hedged, grafted Q. robur tree of mature origin had a positive D value indicating a mature status. Clones initiated from a yearold Q. petraea tree displayed morphology in vitro consistent with a mature status and had a positive D value; however, these clones displayed other traits such as vigor suggesting that vestiges of juvenility remain. Multiplication rate and leaf size and shape were variable among clones and did not provide suitable markers for juvenility or maturity for these Quercus species. Keywords: morphology, nondestructive markers, rejuvenation. Introduction The juvenile phase of shoot development is characterized by annual growth that is rapid and orthotropic, and cuttings have a high rooting potential. The mature phase of shoot development is characterized by slow and plagiotropic growth, and cuttings have a low rooting potential (Meier-Dinkel and Kleinschmit 1990). The characteristics of these phases are of considerable importance for the propagation of elite trees, because it is usually impossible to identify high-quality plants at the seedling stage and difficult to propagate mature trees vegetatively. It is, however, possible to obtain putatively rejuvenated shoots by repeated sequential grafting of adult scions onto juvenile rootstocks, by severe pruning, by application of growth regulators and by in vitro culturing of shoots. Plant material so treated may respond like juvenile tissue, and hence be more receptive to vegetative propagation both in the greenhouse and in vitro (Poethig 1990, Ballester and Meier-Dinkel 1992, Huang et al. 1992a, 1992b, Arnauld et al. 1993, Ke et al. 1993, Ewald and Kretzschmar 1996). To follow the progress of rejuvenation, easily measured qualitative and quantitative criteria of juvenility and maturity that are nondestructive of the shoots are needed; however, such markers are currently lacking in oak and in many other species. Stem diameter and leaf morphology (Lyrene 1981, Brand and Lineberger 1992) and orthotropic or plagiotropic growth (Arnauld et al. 1993) may be used to describe the maturity of the shoot of some species. Rooting potential has been used in several woody plants as a marker for juvenility (Hess 1959, Pliego-Alfaro and Murashige 1987, Huang et al. 1992a). However, in oak, rooting in vitro suffers from a high clonal variability (Meier-Dinkel and Kleinschmit 1990) and shoot tip necrosis (Vieitez et al. 1985), making rooting an unreliable and unsuitable marker for the juvenility status of the donor tree. We have evaluated a range of morphological criteria in shoot cultures from plants of proven juvenile, adolescent ( years old) and mature status to assess their value as indicators of juvenility or maturity. The objective of this study was to characterize mature and juvenile in vitro morphotypes and

2 252 MCGOWRAN, DOUGLAS AND PARKINSON determine if intermediate forms can be identified based on the characteristics of material of known chronological age. Materials and methods Mature and adolescent clones Branch tips from the lower branches of one Quercus robur L. and three Q. petraea ex Liebl. oak trees about 150 years old (Kilmacurragh, Co., Wicklow, Ireland), which were known to be capable of flowering, were collected and grafted to 2-yearold seedlings. New shoots from grafted plants were used to initiate cultures. Branch tips from the crown of a yearold Q. petraea tree, which was known not to be capable of flowering, were grafted to 2-year-old seedlings and new shoots from the grafted plants were used to initiate cultures. A mature clone of Q. robur Fastigiata was initiated from adult budwood of unknown age of severely pruned hedge plants (East Malling, Kent, England) that had been grafted onto 2-year-old seedling rootstocks before culture initiation (Marks and Simpson 1993). Juvenile clones Three juvenile clones of Q. robur were initiated from 7-monthold seedlings (NL3 superior road stand, Veenendaal-de Klomp, Netherlands). Two juvenile clones of Q. petraea were derived from embryos germinated in vitro. Two clones were derived from stump sprouts taken from the base of a 100-year-old Q. robur tree (NL3 superior road stand, Veenendaal-de Klomp) and designated as juvenile because of their origin. Initiation of cultures Closed buds were excised from each plant and sterilized as follows. After a 30-second rinse in 85% ethanol, the buds were immersed in a 0.1% solution of mercuric chloride containing two drops of Tween 20 for 5 min. The buds were then rinsed three times in sterile distilled water (3 min per rinse) followed by a 20-min treatment in calcium hypochlorite (7% solution) and a second series of three rinses in sterile distilled water. Culture conditions Woody Plant Medium (WPM3) (Lloyd and McCown 1980), containing 2.5 g l 1 Phytagel, 20 g l 1 sucrose and 0.2 mg l 1 benzyladenine was used. The ph was adjusted to 5.7 with KOH before autoclaving. All media components were obtained from Sigma Chemicals, Poole, Dorset, England. Medium (32 ml) was dispensed into each 250-ml borosilicate glass jar. After attaching the screw-on aluminum lids, the jars were autoclaved for 20 min at kpa. Nodal segments (1--2 cm long) were placed vertically in each jar. Cultures were grown at 22 ± 2 C in a 16-h photoperiod supplied by warm-white fluorescent lights. Light intensity was approximately 20 µmol m 2 s 1 of photosynthetically active radiation. After 4 weeks of culture, the nodal explants were transferred (five explants per jar) to new medium. Shoot tips and nodal explants ( cm long) that developed on the initial nodal segments were subjected to successive subculturing on WPM3 every 4 to 5 weeks. The nodal explants (2--4 buds per nodal segment) were subcultured horizontally on the medium, whereas the shoot tip explants and the shoots that developed on initial nodes were subcultured by inserting them vertically in the medium. Measurement of morphological characteristics. After a minimum of 18 subcultures, explants were randomly selected and examined 28 days after the most recent subculture. For each clone, a minimum of five explants was examined for morphological characteristics. The angle of the shoot to the horizontal was measured in situ to ± 2 by placing a protractor behind the culture vessel in line with the shoot and horizontal to the medium. Stem length and stem diameters were measured with vernier callipers to ± 20 µm. Stem diameters were measured on internodes immediately above the basal callus, immediately below the whorl of leaves at the shoot tip, and on an internode midway between the basal and tip measurements. Photosynthetic leaves, defined as those that were green, expanded, and firmly attached to the plant, were removed with a scalpel blade, and attached with transparent adhesive tape to white paper and photocopied. The area, breadth, length, perimeter, roundness and aspect ratio of each leaf was then determined by image analysis of the photocopy. An Olympus BX10 TK 1280 E color video camera was used with a Cosmica/Pentax mm TV zoom lens connected to a Quantimet 500 MC Image Processing and Analyzing system with associated software (Leica Cambridge Ltd., Cambridge, England). Roundness was defined as (Perimeter) 2 /(4π Area 1.064). Scale leaves, defined as brown, small (< 2 mm in length) and easily detached from the shoot, were counted. The number of shoots (> 1 mm in length) arising from each cultured explant was also recorded. Multiplication rate was defined as the number of explants obtained per viable subcultured explant. Statistical analysis All statistical analyses were carried out with SPSS for windows (SPSS, Inc., Chicago, IL). One-way analysis of variance was carried out on shoot cultures of juvenile and mature origin. For discriminant analysis, the shoots were divided into those of seedling and stump sprouts origin, and those derived from mature, flowering trees, excluding shoots derived from year-old trees, and those of the severely hedged stock plant. All factors (shoot angle, mid, base and stem tip diameter, number of green leaves, number of scale leaves, number of shoots, and shoot length) were included. Discriminant analysis was carried out on the data by a stepwise selection of factors using Wilks Lambda. Results Shoot morphology Morphological characteristics of shoot cultures derived from seedling, stump sprout and mature clones of Q. robur are summarized in Table 1. Results for shoot cultures derived from seedlings, year-old trees and mature flowering and TREE PHYSIOLOGY VOLUME 18, 1998

3 MARKERS OF JUVENILITY AND MATURITY IN QUERCUS SPP. 253 hedged stock plants of Q. petraea are summarized in Table 2. For Q. robur, the characteristics of the two clones derived from stump sprouts were similar to those of the three clones derived from seedlings and these five clones could be distinguished from cultures derived from mature trees and hedged stock plants by angle of shoot growth and by stem-tip and mid-stem diameter. Similarly, two seedling-derived clones of Q. petraea were distinguishable from three clones derived from mature trees by these same morphological traits. Cultures derived from a year-old (adolescent) tree of Q. petraea exhibited morphological characteristics that were more similar to mature clones than to juvenile material. The cultures could therefore be grouped according to the maturity of the initial explants. A comparison of all clones with juvenile characteristics (five Q. robur and two Q. petraea) with those showing mature characteristics (two Q. robur, four Q. petraea) revealed that the angle of shoot growth to the horizontal was greater for clones derived from seedling and stump sprouts than for clones derived from mature or year-old trees. Similarly, clones derived from seedlings and stump sprouts had smaller stem-tip diameters and mid-stem diameters than clones derived from mature or year-old trees, except for one mature clone of Q. petraea. The number of green leaves per shoot, number of scale leaves per shoot, stem base diameter and shoot length were highly variable among clones for both oak species and there were no striking differences in these characteristics between the seedling and stump sprout clones and the mature and adolescent clones. The number of shoots per explant tended to be higher for the seedling and stump sprout clones than for the adolescent and mature clones; however, it was highly variable. Scale leaf number was difficult to assess in both species because of the tendency of scale leaf material to detach easily from the explant stems, which may have contributed to the variability in this characteristic. In all clones except the hedged stock plants, the diameter at the base of the stem was generally greater than elsewhere on the shoot. One-way ANOVA on juvenility status was carried out on all raw data for Q. petraea and Q. robur clones with the exception of the Q. robur clone derived from hedged stock plants which could not be assigned unambiguously to either juvenile or mature categories. In the case of Q. petraea, highly significant differences were obtained for angle of shoot to horizontal (P = ), stem-tip diameter (P = ), and mid-stem diameter (P = ) between juvenile and mature explants. The ANOVA of Q. robur clones indicated a highly significant difference in mid-stem diameter (P = ) and stem-tip diameter (P = ) between clones designated as juvenile (seedling and stump sprout origin) or mature (mature flowering). Angle of shoot to the horizontal marginally failed to achieve significance (P = ). Discriminant analysis was carried out on raw data for Q. petraea and Q. robur clones, excluding the clone derived from hedged stock plants. The analysis yielded coefficients associ- Table 1. Morphological characteristics of in vitro shoot cultures of Quercus robur derived from seedlings, stump sprouts, mature crown scions on grafted plants and old hedged stock plants. Measurements were made 4 weeks after the final subculture. Values are shown with the standard error of the mean; n = explant number for each clone. Origin of clone n Shoot angle No. green No. scale Shoot tip Mid-stem Stem base Shoots per Shoot to horizontal leaves leaves per diameter diameter diameter explant length ( ) per shoot shoot (mm) (mm) (mm) (mm) Seedling ± ± ± ± ± ± ± ± 3.00 Seedling ± ± ± ± ± ± ± ± 5.21 Seedling ± ± ± ± ± ± ± ± 2.76 Stump sprouts ± ± ± ± ± ± ± ± 5.79 Stump sprouts ± ± ± ± ± ± ± ± 5.81 Mature flowering ± ± ± ± ± ± ± ± 4.60 Hedged stockplants ± ± ± ± ± ± ± ± 2.84 Table 2. Morphological characteristics of in vitro shoot cultures of Quercus petraea derived from seedlings, scions from a year-old tree grafted to seedlings (adolescent) and scions from a 150-year-old tree grafted to seedlings (mature). Measurements were made 4 weeks after the final subculture. Values are shown with the standard error of the mean; n = explant number for each clone. Origin of clone n Shoot angle No. green No. scale Shoot tip Mid-stem Stem base Shoots per Shoot to horizontal leaves leaves per diameter diameter diameter explant length ( ) per shoot shoot (mm) (mm) (mm) (mm) Seedling ± ± ± ± ± ± ± ± 3.19 Seedling ± ± ± ± ± ± ± ± 3.64 Adolescent ± ± ± ± ± ± ± ± 1.85 Mature flowering ± ± ± ± ± ± ± ± 2.50 Mature flowering ± ± ± ± ± ± ± ± 1.24 Mature flowering ± ± ± ± ± ± ± ± 2.00 TREE PHYSIOLOGY ON-LINE at

4 254 MCGOWRAN, DOUGLAS AND PARKINSON ated with morphological traits that allow us to propose the following formulae to assign a discriminant score (D) to the Q. petraea and Q. robur clones. For Q. petraea: D = SA TD MSD, (1) where SA is stem angle ( ), TD is tip diameter (mm) and MSD is mid-stem diameter (mm). For Q. robur: D = TD MSD. (2) Negative discriminant score values indicate that the material is juvenile, whereas positive values indicate that the material is mature. Discriminant scores for clones of Q. robur and Q. petraea are shown in Figures 1 and 2, respectively. For Q. robur, all clones of seedling origin or derived from stump sprouts were closely grouped with an average negative discriminant score. The mature Q. robur clone derived from a mature tree had a strongly positive discriminant score. Among the clones, the clone derived from the hedged stock plants had the highest D value. For Q. petraea, both clones of seedling origin were closely grouped with an average negative discriminant score. All clones of mature or adolescent origin were closely grouped with an average positive discriminant score. The yearold (adolescent) clone scored strongly positively indicating a mature status, and suggesting that the remaining morphological characters were mature, even though the tree had not yet flowered. Leaf morphology Image analysis of leaf material (Table 3) indicated differences within species between mature and juvenile clones in surface Figure 2. Discriminant scores for Quercus petraea clones. All values are the mean of at least six replicates and are shown with the standard error of the mean. Table 3. Leaf characteristics for juvenile and mature Quercus robur and Q. petraea. Measurements for juvenile Q. robur are based on 268 leaves and those for mature Q. robur are based on 46 leaves. Measurements for juvenile Q. petraea are based on 182 leaves and those for mature Q. petraea are based on 266 leaves. Roundness = (Perimeter) 2 /(4π Area 1.064). Leaf character and Quercus petraea Quercus robur maturity state Mean ± SE Mean ± SE Area (mm 2 ) Juvenile ± ± 2.62 Mature ± ± 7.81 Length/width Juvenile 2.36 ± ± 0.03 Mature 2.38 ± ± 0.06 Width (mm) Juvenile 6.06 ± ± 0.17 Mature 4.27 ± ± 0.41 Length (mm) Juvenile ± ± 0.48 Mature 9.78 ± ± 0.96 Perimeter (mm) Juvenile ± ± 1.46 Mature ± ± 2.47 Roundness Juvenile 1.70 ± ± 0.09 Mature 1.80 ± ± 0.04 Figure 1. Discriminant scores for Quercus robur clones. All values are the mean of at least five replicates and are shown with the standard error of the mean. area, perimeter, lamina length and width of leaves. For Q. robur clones, surface area, width, perimeter and lamina length of leaves were all greater in the mature clones than in the juvenile clones, whereas for Q. petraea clones, surface area, width, perimeter and laminar length of leaves were all greater in the juvenile clones. These initial findings suggest a possible means to distinguish between Q. robur and Q. petraea clones grown in vitro based on leaf characteristics. However, TREE PHYSIOLOGY VOLUME 18, 1998

5 MARKERS OF JUVENILITY AND MATURITY IN QUERCUS SPP. 255 the results are based on large numbers of leaves (452 leaves from juvenile shoots and 312 leaves from mature shoots) and the technique may therefore not be useful for routine monitoring of shoot cultures. Furthermore, there was wide variability within each measured parameter. Moreover, neither roundness nor aspect ratio differed significantly between leaves from clones of juvenile or mature origin, suggesting that there may be only subtle changes in the shape of the leaf with maturity. Discriminant analysis of the leaf data indicated a considerable overlap between juvenile and mature leaf morphology. Multiplication rates Based on the mean multiplication rate obtained over 16 culture cycles, juvenile clones tended to have a higher multiplication rate than mature clones, especially for Q. robur (Table 4). However, there was high variability in the multiplication rate both between clones of a similar maturity state and between subcultures within a clone (results not shown), indicating that this trait was not a reliable indicator of maturity status. Discussion Morphological characteristics of shoots from in vitro cultures may be used to differentiate juvenility and maturity in both Q. robur and Q. petraea. For Q. petraea, shoot angle to the horizontal was the most useful and least variable of the parameters. For Q. robur clones, stem-tip diameter and mid-stem diameter were the most useful criteria. Plagiotropic growth was observed in vitro for both species and differed significantly between juvenile and mature states only for Q. petraea. These results are consistent with those found for other woody plant species (Brand and Lineberger 1992, Arnauld et al. 1993). Two Q. robur clones that originated from stump sprouts of a 100-year-old Q. robur had in vitro growth characteristics suggestive of juvenile status (shoot production and vigor). A quantitative analysis of the traits of both clones yielded a negative discriminant score, indicative of a juvenile status. This is consistent with the belief that stump sprouts have some Table 4. Multiplication rates of Quercus cultures after 16 subculture cycles. Clone No. of Mean no. of explants explants produced per explant subcultured ± SE Quercus robur Seedling ± 0.12 Seedling ± 0.06 Stump sprouts ± 0.11 Hedged stockplant ± 0.19 Quercus petraea Seedling ± 0.07 Seedling ± 0.38 Adolescent ± 0.10 Mature flowering ± 0.14 juvenile morphological characteristics (Hackett 1985, Franclet et al. 1987). Severe pruning has been used as a means of rejuvenation in several plant species (Hatcher 1959, Garner and Hatcher 1962, Black 1972, Franclet 1979). However, the clone of Q. robur derived from a severely pruned hedge displayed a highly significant difference in stem diameter when compared to juvenile material, indicating a mature status. Marks and Simpson (1993) also noted that this clone displayed qualitative characteristics typical of mature clones. These observations support Hackett s (1985) view that severe pruning is unlikely to rejuvenate plants fully. Full rejuvenation of material did not arise from grafting of mature shoots because our mature clones and the yearold clone were derived from grafted plants and all of these clones displayed mature characteristics in vitro. The discriminant score obtained for different juvenile or mature clones was variable for both species, indicating that genotype may be an important factor. In the case of mature clones, the rootstock may have imparted different rejuvenating effects. However, in vitro culturing per se of mature clones was not a sufficient treatment to reverse the maturity status. It may be possible to quantify the progressive effects of rejuvenation treatments before culturing or during in vitro culture by monitoring the effects of treatment and culture times on the value of D. In oak species, the length of the juvenile period is estimated to be about years (Clark 1983). Shoot culture of Q. petraea derived from a year-old non-flowering tree gave a positive discriminant score suggesting a mature physiological status. In addition, shoot angle to the horizontal, length of shoots and shoot tip diameter were consistent with those for mature material. However, unlike three clones of Q. petraea derived from 150-year-old trees grafted to 2-year-old seedlings, the year-old clone was not difficult to culture, did not display recalcitrant characteristics such as phenolic exudation, and produced vigorous new shoots. Because the year-old clone had not flowered and flowering is the true indicator of sexual maturity, our results suggest that morphological maturity precedes sexual maturity and that shoot vigor is a good indicator of the transition period between morphological maturity and sexual maturity. All of the seedling-derived clones showed shoot vigor similar to that of our year-old clone, indicating that the main morphological maturation processes occurred within a period of about years in oaks. We speculate that shoot vigor in combination with discriminant analysis could provide a means of identifying intermediate maturity morphotypes. Neither multiplication rate nor morphology of leaves from in vitro shoot cultures provided a suitable means to differentiate between juvenile and mature material because both parameters were highly variable. Other authors have used leaf characters of field-grown trees to distinguish Q. petraea and Q. robur (Rushton 1983, Kleinschmit et al. 1997), suggesting that it may be possible to confirm differentiating traits between species and states of maturity. The use of image analysis and the advent of more advanced software offers a powerful tool TREE PHYSIOLOGY ON-LINE at

6 256 MCGOWRAN, DOUGLAS AND PARKINSON for the morphological assessment of leaf material (Adams and Thomas 1987). It is also nondestructive of the shoots. We conclude that the developmental age of donor trees strongly affects the vigor and morphological traits of in vitro cultured shoots of Q. robur and Q. petraea. For both species, there was a tendency to thicker stems and plagiotropic growth in shoots derived from mature clones. Despite the inherent variability in morphological characteristics, there were significant differences between juvenile and mature shoots to permit us to define juvenility status of the shoot in terms of a single parameter that can be easily quantified. These morphological markers are therefore potentially useful for monitoring growth and development in vitro in terms of developmental status and for quantifying the effects of treatments such as repeated subculturing and serial grafting on the recovery of more juvenile characteristics. Our aim is to reinforce these morphological markers with complementary parameters. There is evidence that biochemical markers (Bonn 1988, Murray et al. 1991, Amo-Marco et al. 1993, Murray et al. 1994, Woo et al. 1994) may be a useful supplement. We are currently examining a range of biochemical markers for identifying juvenility and maturity in oak cultures. Acknowledgments This work was funded in part by a Forbairt Grant (No. SC\93\062) to E. McGowran and an EC Eclair Contract to G.C. Douglas. The authors thank Dr. Tim Marks of Horticultural Research International, England, for providing shoot cultures from a severely pruned hedge, and Dr. Eva Waindinger, Forschungszentrum Seibersdorf, Austria for the donation of shoot cultures of juvenile Q. petraea. Grateful thanks to Dr. Padraig Walsh and Mr. Donal O Shea of Dublin City University for assistance in image analysis. References Adams, H.L. and C.R. Thomas The use of image analysis for morphological measurements on filamentous microorganisms. Biotechnol. Bioeng. 32: Amo-Marco, J.B, A. Ballester, N. Vidal and A.M. Vieitez Polypeptide markers differentiating juvenile and adult tissue in chestnut. J. Plant Physiol. 142: Arnauld, Y., A. Franclet, H. Tranvan and M. Jacques Micropropagation and rejuvenation of Sequoia sempervirens (Lamb.) Endl.: a review. Ann. Sci. For. 50: Ballester, A. and A. Meier-Dinkel Micropropagation of Quercus species. In Micropropagation of Betula and Quercus: Initial reports of the COST 87 Woody Plant Working Group. Ed. F. O Riordan. Comm. European Communities, DG XII, Brussels, pp Black, D.K The influence of shoot origin on the rooting of Douglas-fir stem cuttings. Proc. Int. Plant Propag. Soc. 22: Bonn, M.C J16: an apex protein associated with juvenility of Sequoiadendron giganteum. Tree Physiol. 4: Brand, M.H. and R.D. Lineberger In vitro rejuvenation of Betula (Betulaceae): Morphological evaluations. Am. J. Bot. 79: Clark, J.R Age-related changes in trees. J. Aboric. 9: Ewald, D. and U. Kretzschmar The influence of micrografting in vitro on tissue culture behaviour and vegatative propagation of old European larch trees. Plant Cell Tissue Organ Cult. 44: Franclet, A., M. Boulay, F. Bekkaoui, Y. Fouret, B. Verschoore-Martouzet and N. Walker Rejuvenation. In Cell and Tissue Culture in Forestry, General Principles in Biotechnology, Vol. 1. Eds. J.M. Bonga and D.J. Durzan. Martinus Nijhoff Publishers, Dordrecht, The Netherlands, pp Franclet, A Rajeunissement des arbres adults en vue de leur propagation végétative. In Micropropagation d Arbres Forestiers. AFOCEL, France, Études et Recherches No. 12-6/79: Garner, R.J. and E.S.J. Hatcher Regeneration in relation to vegetative vigor and flowering. Proc. 16th Int. Hortic. Congr., Brussels, pp Hackett,W.P Juvenility, maturation, and rejuvenation in woody plants. Hortic. Rev. 7: Hatcher, E.J.S The propagation of rootstocks from stem cuttings. Ann. Appl. Biol. 47: Hess, C.E A study of plant growth substances in easy and difficult to root cuttings. Proc. Int. Plant Propag. Soc. 9: Huang, L.C., C. Hsiao, S. Lee, B. Huang and T. Murashige. 1992a. Restoration of vigor and rooting competence in stem tissue of mature citrus by repeated grafting of their shoot apices onto freshly germinated seedlings in vitro. In Vitro Cell Dev. Biol. 28: Huang, L.-C., S. Lius, B.-L. Huang, T. Murashige, E.F.M. Mahdi and R. Van Gundy. 1992b. Rejuvenation of Sequioa sempiverens by repeated grafting of shoot tips onto juvenile rootstocks in vitro. Plant. Physiol. 98: Ke, S., Q. Cai and R.H. Shirvin Micrografting speeds growth and fruiting of protoplast-derived clones of Kiwifruit (Actinidia deliciosa). J. Hortic. Sci. 68: Kleinschmit, J.R.G., R. Bacilieri, R. Kremer and A. Roloff Comparison of morphological and genetic traits of pedunculate oak (Q. robur L.) and sessile oak (Q. petraea (Matt) Liebl.) Silvae Genet. 44: Lloyd, G. and B.H. McCown Commercially feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot tip culture. Proc. Int. Plant Propag. Soc. 30: Lyrene, P.M Juvenility and production of fast-rooting cuttings from blueberry shoot cultures. J. Am. Soc. Hortic. Sci. 106: Marks, T.R. and S.E. Simpson Changes in the competence of Quercus robur Fastigiata to grow in vitro as affected by seedling rootstocks and differential pruning. J. Hortic. Sci. 68: Meier-Dinkel, A. and J. Kleinschmit Aging in tree species: Present knowledge. In Plant Aging: Basic and Applied Approaches. Eds. R.R. Rodriguez, T. Sanchez and D.J.Durzan. Plenum Press, New York, pp Murray, J.R., A.G. Smith and W.P. Hackett Dihydroflavonol reductase activity in relation to differential anthocyanin accumulation in juvenile and mature phase Hedera helix L. Plant Physiol. 97: Murray, J.R., A.G. Smith and W.P. Hackett Differential dihydroflavonol reductase transcription and anthocyanin pigmentation in the juvenile and mature phases of ivy (Hedera helix). Planta 194: Pliego-Alfaro, F. and T. Murashige Possible rejuvenation of adult avocado by graftage onto juvenile rootstocks in vitro. Hortscience 22: Poethig, R.S Phase change and the regulation of shoot morphogenesis in plants. Science 250: TREE PHYSIOLOGY VOLUME 18, 1998

7 MARKERS OF JUVENILITY AND MATURITY IN QUERCUS SPP. 257 Rushton, B.S An analysis of variation of leaf characters in Quercus robur L. and Quercus petraea (Matt.) Liebl. population samples from Northern Ireland. Irish For. 40: Vieitez, A.M., M.C. San Jose and E. Vieitez In vitro plantlet regeneration from juvenile and mature Quercus robur L. J. Hortic. Sci. 60: Woo, H.-H., W.P. Hackett and A. Das Differential expression of a chlorophyll a/b binding protein gene and a proline rich protein gene in juvenile and mature phase English ivy (Hedera helix). Physiol. Plant. 92: TREE PHYSIOLOGY ON-LINE at

8