Báo cáo khoa học: "Rejuvenation of Quercus robur"

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article Original of Quercus robur Rejuvenation P Evers E Vermeer, S Eeden van IBN-DLO, Institute for Forestry and Nature Research, Department of Urban Ecology, Section of Ecophysiology, POBox 23, 6700 AA Wageningen, The Netherlands Summary — Stem sections and some branches, 30 cm in length, of mature Quercus were cut to in- duce the formation of rejuvenated shoots as initial material for in vitro propagation. Up to 100-year- old trees were used and topophysical effects were taken into account. In vitro rooting of shoot tips taken from this material showed a lower efficiency than embryo-derived cultures, but the fact that rooting occurred indicates some degree of rejuvenation. The influence of stem topophysis as well as genotype remains unclear. rejuvenation / Quercus robur / topophysis / adventitious neoformation Résumé — Réjuvénilisation de Quercus robur. Des segments de la tige principale ainsi que des branches ont été prélevés sur des chênes âgés de manière à induire la formation de pousses reju- vénilisées destinées à la multiplication in vitro. Des arbres de plus de 100 ans ont été retenus et les effets de topophysis ont été pris en compte. L’enracinement in vitro de ces pousses est plus difficile que celui produit dans des cultures d’embryons. Mais il indique toutefois un certain degré de rejuvé- nitisation. L’influence de la topophysis et du génotype de l’arbre n’a pas été élucidée. réjuvénilisation / Quercus robur / topophysis / néoformation adventive INTRODUCTION have therefore been proposed before (eg, Romberger, 1976) but were not so often effectuated. Also in Quercus robur, this Micropropagation of woody species has difference was observed and some rejuve- been reported to be limited by rootability in nation attempts implemented (Chalupa, many surveys (eg, Bonga and Durzan, 1984; Meier-Dinkel, 1987; Vermeer and 1987) of shoot tips harvested from adult Evers, 1987; Evers et al, 1988; Ballester trees. Embryo-derived cultures, used as et al, 1990). In Dutch forestry, this species the juvenile reference, in general proved is used both for urban and planted stand to be more efficient in growth rate, axillary purposes. For urban areas, selected elite branching and rooting than adult tree- genotypes are used, usually still propagat- derived cultures. Rejuvenation strategies ed through grafting. For stands, acorns
From the 65-year-olds, stem and branch sec- harvested from selected seed stands. are tions were taken. All of the 8-year-old and two- means that the development of micro- This thirds of the 100-year-old sections were taken to propagation techniques starting from both the greenhouse due to a lack of space; for the adult and juvenile material are required 65-year olds, 3 sections out of each of the total and, at the same time, provide the oppor- number of sections present in each fourth por- tunity to study the consequences of matu- tion of the stem height were randomly selected. This reduction was again due to the lack of ration. Maturation is often associated with greenhouse space. the loss of rootability and with the success of promotion of flowering (Libby, 1974). comparison For the 65-year-olds, was a made between 6 genotypes that form epicormic The results presented in this paper reflect shoots (defined as any stem shoot occurring in the attempts to attain rejuvenation through intact trees) readily and those that do not. In the application of some techniques on the the regenerated or activated shoots from the mother trees followed by micropropaga- sections, usually occurring within 9 days, a dis- tion. The rooting results are compared with tinction was made between shoots presumably those of the embryo-derived material. The of sphaeroblastic (adventitious) origin and shoots of accessory origin (axillary shoots oc- possibility that the applied techniques curring near branch scars). The average quar- have led to the formation of adventitious ter height part of the stem contained an aver- buds (shoots of epicormic and/or sphaero- age of 17 sections. One of every 3 sections blastic origin) in the mother trees is dis- was incubated in the greenhouse. The 5 stem cussed. height sections of the 100-year-old trees were defined simply by dividing the height into 5 parts. Some of the 100-year-old trees in the same stand were severely branch-pruned in MATERIALS AND METHODS situ to induce shoot formation in the field. All mechanical pretreatments were done in March. Acorns were harvested from the Ede/de Klomp Shoot tips were cultured on WPM with 20 g/l seed stand. Embryos were pulse treated with activated charcoal which was lowered to 5 g/l 0.5 mM 6-benzyladenine (BA) prior to culture on in the next subculture. The regulators were: 6- woody plant medium (WPM). Rooting was done BA, 4.4 μM in the initial phase and 2.2 μM in on WPM with 4.9 μM indole-3-butyric acid (IBA) the multiplication phases thereafter; for rooting and 8 g/l activated charcoal for 4 weeks. In ear- (attempted from the 5th in vitro cycle onwards lier research, increased sucrose levels proved on different macromedia), IBA at 4.6 μM with- to inhibit the effect of IBA and, therefore, were out activated charcoal was used. Plantlets not used. Adult trees were 8-, 65- and 100- were planted out in potting soil and gradually years old respectively; the 8-year-old trees were adapted to the lower humidity in the green- defined as ’adult’ because in vivo rooting was house by opening the plastic tent. no longer possible but, if the ability to induce flowering is used as a criterion, this stage had not yet been reached. RESULTS From the 8- and 100-year-old trees, stem sections of 30 cm in height were sawed in March and incubated upright in peat-potting soil The reference material, embryo-derived for one-third of their length in the greenhouse shoot clusters showed a multiplication rate after being disinfected with 1% Captan. For this of 3.4-3.7 (Standard error of difference of purpose, the trees were cut down, leaving a 30- cm stump remaining. Stem and branch sections means 2.4) after 3 subcultures. The aver- were sawed on the forest floor. The stem sec- age rooting efficiency in vitro depends tions, intact lengths of the stem, were incubated upon the method (especially the sucrose in a plastic tent to maintain high humidity. The concentration when IBA is not applied) and cut surfaces did not receive any further treat- genotype, but can be as high as 100% on ment. Shoot tips were harvested from one of the a medium with 30 g/l sucrose. remaining stumps in the field 8 weeks later.
The results of shoot regeneration in vivo experiments, branch shoot regeneration showed a large variation between the seg- showed comparable results, especially ments and the genotypes. The segment when the bark surface area was taken into shoot production of the different mother account. Shoots from the in situ treatments tree according to age was: 195 for 8-year- (telegraph post, branch) were not counted old trees (whole stem in 13 segments, all and were impossible to isolate in vitro due used), 240 for 65-year-old trees (1/3 of the to contamination problems. segments used, estimated 720 for the The data obtained from the 100-year- whole tree) and 893 for 100-year-old trees old (figs 1, 2), 65-year-old (fig 3) and 8- (2/3 of the segments used, estimated 1340 year-old trees showed that no clear stem for the whole tree). In the 100-year-old topophysical effects in regenerating adven- trees, 21% of the shoots may be adventi- titious or axillary shoots were apparent. Of tious, since they did not occur close to the average total number of shoots regen- branch scars; for the 65-year-olds and 8- erated on segments from 65-year-old year-olds the percentages were 1.6 and trees, 55% were found in the lower half of 35%, respectively. In the 65-year-olds, the the stem, a part presumed to be more difference between the epicormic-rich and juvenile as a result of ontogenesis. Howev- epicormic poor was 393 and 87, respec- er, in the epicormic-rich group, this per- tively. The number of presumably adventi- centage was 63%, while in the epicormic- tious origin on trees of the 3 ages from old- poor group it was only 16%. est to youngest were: 68, 4 and 183, The juvenility parameter ’maximum in vi- respectively, again on the above men- rooting’ did not differ significantly be- tro tioned portion of segments. In preliminary
sider the ability to flower as transition to material tween the (100% embryo-derived the adult stage; in that sense, 8-year-olds maximum, 72% average) and 8-year-old- are not yet adult. It is however clear that, derived material (93% maximum, 87% av- with increasing age of the mother tree, The percentage in the old trees is erage). shoot material is regenerated that is in present < 20% on the average, but is ex- at terms of in vitro behavior not like the pected to go up once the in vitro age of shoots derived from embryo cultures. This this material increases. In some experi- apparently incomplete rejuvenation may be ments, it was as high as 37%, especially full rejuvenation (comparable to germinat- when regenerants from the most basal root ing seedlings), because it can be obscured part of stumps from the forest were used. by non-optimal in vitro conditions causing multiplication rate of the 8-year-olds The a more ’adult’ type of morphogenesis. not lower than that of embryo-derived was Also, expression of rejuvenation may de- material (4.0 vs 3.4 mo). The 65-year-old pend upon morphogenesis. Also, expres- trees were not in culture long enough to sion of rejuvenation may depend upon in give conclusive results. The highest multi- vitro age: it is a well-known phenomenon plication rates (5.4, 100-year-old trees) that material coming from older trees has a were found in shoot-tip material in vitro slower ’acceleration’ in initial morphogene- harvested from regenerates on the basal sis. On the other hand, it may simply be a part of the main roots of felled trees; the changing metabolism, even in flushing lowest rate occurred in tips from accessory resting buds that were not formed in the buds from the highest part of the stem (no previous season (Libby, 1974). multiplication at all). From 2 model 100- theories of Nozeran et al Based on year-old trees, a total of 858 and 466 shoot (1971),the observed rejuvenation could tips for tissue culture could be harvested also be a topophysically based difference (2/3 of the segments). in the level of differentiation. Our earlier re- Acclimatization in the greenhouse of 8- sults indicated a relationship between in vi- year-old-derived material did not differ from tro morphogenesis and mother plant to- that of the embryo-derived plantlets. How- pophysis (Evers, 1984; Evers et al, 1988). ever, growth in the greenhouse and the This hypothesis, considering the lack in nursery of plantlets derived from the 8- stem segment discrepancies in the results much slower. year-old material was presented in this paper, seems not to be the case. According to Halle et al (1978), the observed regeneration might also be DISCUSSION called reiteration, indicating a more adven- titious nature. The definitions given in the literature for regenerating stem and branch principle of rejuvenation in Q robur The shoots are very unclear in terms of being has been proven, if rooting in vitro is used adventitious or not. This distinction is of ut- as a parameter. The in vivo reference, the most importance to be able to estimate ability to root crown branch cuttings was risks of the occurrence of genetic aberra- not successful. The in vitro reference, em- tions. Histological studies may yield more bryo-derived shoot tips, proved to be no conclusive results in the near future. more efficient than the tips from 8-year-old segment-derived material. The question is The results for the 65-year-olds may in- whether 8-year-old trees can be called dicate a more discouraging aspect: a rela- adult or whether they are in a transition tionship between epicormic-rich geno- types, less desirable for many applications, phase from juvenility. Some scientists con-
Evers PW, Donkers J, Prat A, Vermeer E (1988) and the ability to regenerate stem and Micropropagation of forest trees through tis- branch shoots from segments. The geno- sue culture. Pudoc Wageningen 115 p typic differences are not well understood. Halle F, Oldeman RAA, Tomlinson PB (1978) Some of the differences thought to be gen- Tropical Trees and Forests. Springer, Berlin otypic may, in the end, turn out to be Libby WJ (1974) The use of vegetative propa- growth-site-induced in comparable geno- gules in forest genetics and tree improve- types. This last factor will be a major item ment. N Z J For Sci 4, 290-296 in our research. Meier-Dinkel A (1987) In vitro Vermehrung und Weiterkultur von Stieleiche (Quercus robur) und Traubeiche (Quercus petraea). Allg Forst REFERENCES Jagdztg 158, 199-204 Nozeran R, Bancilhon L, Neville P (1971) Inter- Ballester A, Sanchez C, San-José MC, Vieitez vention of internal correlations in the morpho- FC, Vieitez AM (1990) Development of reju- genesis in higher plants. In: Advances in venation methods for in vitro establishment. Morphogenesis (Abercombie A, Brachet J, In: Plant Ageing: Basic and Applied Ap- King TJ, eds) Academic Press, London, 1-66 proaches (Rodriguez R, Durzan D, Sanchez JA (1976) An appraisal of prospects Romberger R, eds) Plenum Press, New York, 214-218 for research on juvenility in woody perenni- Durzan DJ (1987) Cell and Tissue Bonga JM, als. Acta Hortic Wag 56, 301-317 Culture in Forestry. Nijhoff, Dordrecht Vermeer E, Evers PW (1987) Induction of Chalupa V (1984) In vitro propagation of oak branching and nutrient replenishment in em- (Quercus robur) and Linden (Tilia cordata). bryo culture of Quercus robur. In: Florizel, 374-377 Biol Plant 26, Plant Micropropagation in Horticultural Indus- Evers PW (1984) Growth and morphogenesis of tries (Ducote G, Jacob M, Simeon A, eds) Douglas fir in vitro. Pudoc Wageningen, 110 p Presses Universitaires, Liege, 345-347