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Review article Micropropagation and rejuvenation of Sequoia sempervirens (Lamb) Endl: a review Y Arnaud A Franclet H Tranvan M Jacques 1 Université P et M Curie, Paris VI, Laboratoire de Physiologie du Développement des Plantes, Tour 53, 75252 Paris Cedex 05; 2 Institut de Recherche Agronomique et Forestière, 13 avenue de Champabon, 77520 Donnemarie-Dontilly, France 5 17 December (Received May 1992; accepted 1992) Summary — This article describes the botanical, biological and forest-tree characteristics of Se- quoia sempervirens, the reasons for interest in its in vitro vegetative multiplication, the difficulty in achieving this from old and remarkable trees, and reviews means of overcoming this limitation. Among such means are the repeated culture of stem fragments on media containing appropriate hormonal combinations, the micrografting of buds originating from old trees onto juvenile rootstocks, and regeneration of buds from previously rejuvenated material. The value and limitations of these protocols and of morphological, physiological and biochemical markers of rejuvenation are dis- cussed. The experimental conditions required for the formation of somatic embryos are described. Increased knowledge of in vitro micropropagation will be essential to enhance the use of clonal se- lection and offer practical outlets to studies concerning somatic hybridization and somatic embryo- genesis. micropropagation / rejuvenation / Sequoia sempervirens I somatic embryogenesis Résumé — Micropropagation et rajeunissement du Sequoia sempervirens (Lamb) Endl : Cet article présente les principales caractéristiques botaniques, biologiques et forestières du revue. Sequoia sempervirens. Il analyse l’intérêt de la multiplication végétative réalisée in vitro soit par mi- cropropagation sensu stricto (figs 1, 2 et 3), soit par régénération (figs 6, 7 et 9); il discute les rai- sons de la difficulté à la réaliser à partir d’arbres âgés et remarquables ainsi que les moyens de la contourner. Parmi ces moyens figurent la réitération des cultures de fragments de tige sur des mi- lieux contenant un équilibre hormonal adéquat (tableaux I et II), le microgreffage de bourgeons ap- partenant à des plantes âgées sur des porte-greffes juvéniles (figs 4 et 5), la régénération de bour- geons à partir de matériel préalablement rajeuni selon l’un des protocoles précédents (figs 10 et 11). L’intérêt et les limites de ces protocoles sont discutés, en considérant les marqueurs morphologi- ques (figs 12 et 13), physiologiques et biochimiques du rajeunissement. Les conditions d’obtention de l’embryogenèse somatique chez cet arbre sont décrites (fig 8). Finalement, l’accroissement de nos connaissances en micropropagation in vitro apparaît essentiel pour augmenter la qualité de la sélection clonale et offrir des débouchés pratiques aux travaux concernant l’hybridation somatique et l’embryogenèse somatique. L’acquisition de telles connaissances de base devrait permettre une meilleure utilisation de cet arbre. embryogenèse somatique / micropropagation / rajeunissement / Sequoia sempervirens
INTRODUCTION pits, frequently biserial or triserial (Jacqui- ot, 1955). Lacking resinous vessels, the heart-wood is particularly fire-resistant Sequoia sempervirens (Lamb) Endl is dis- when mature. The long fibers (= 4 mm) in tinguished not only by its exceptional vig- the heart-wood represent 70 to 90% of its our and long life, but also by its productivi- dry weight (Donnet, 1984). The sapwood is ty of quality wood. It is a gymnosperm white and homogeneous (Rol, 1981). belonging to the Coniferophytes, order Cu- Sequoia sempervirens is a heteroplastic pressales, family Taxodiaceae (Stokey, species. In very young trees, the long axis 1981).Itis the only species of the genus (long twig) generally bears acicular leaves Sequoia (Chadefaud and Emberger, with an axial phyllotaxy. The short axes 1960). The number of chromosomes is (lateral twigs) bear light-green, wide and 2n = 66 (Stebbins, 1948), with 1 to 6 ac- soft leaves, attached at a right, or nearly cessory chromosomes, according to the right angle on the axis, with crosswise- population (Libby and McCutchan, 1978). opposite phyllotaxy. Several cultivars have been registered and commercialized (Redher, 1958; Chaudun, In adult or old trees the long, soft, light- 1977). green leaves of the stump sprouts and suckers are similar to those issued from seedlings. In the lower part of the crown, Origin and geographical area the long and short twigs resemble those of young trees. In the upper part of the crown, the long twigs have squamiform The current genus Sequoia is said to de- leaves which, in very old trees, look like rive from an ancestral complex, a compo- those of Sequoiadendron; the short twigs nent of which is the genus Rhombostrobus are feather-like, with the leathery awl- (upper Cretaceous) (LaPasha and Miller, shaped, dark green (sometimes bluish 1981).Sequoia langsdorfii, a fossil species green leaves), which form an almost acute presenting narrow parental links with the angle with the axis of the twig. current Sequoia sempervirens, is sup- In the northern hemisphere Redwood posed to have disappeared from Europe at blooms between November and early the end of the tertiary era (Emberger, March (Boulay, 1989). The monosporan- 1968). giate inflorescences are in a terminal posi- The natural of extant Sequoia sem- area tion on the short twigs (Debazac, 1964). pervirens extends from southwest Oregon Male cones (< 10 mm long) can also be in to California (USA). In the 19th century axillary positions and possess numerous some specimens were exported to Russia, stamens. The female cones are 20 to Great Britain and France (Donnet, 1984). 25 mm long, with 15 to 20 woody scales. The seeds weigh on average 4 mg and are brown, elliptical and bordered by a small Morphology and development wing. Germination rate is very variable. Many seeds are often empty, the embryos Sequoia sempervirens is an evergreen badly formed or infected by parasites. Vi- species, with a thick (up to 25 cm), fibrous, able seeds are stored with difficulty (Don- deeply furrowed bark. The heart-wood is net, 1984; Bourgkard and Favre, 1989). reddish brown (hence the name ’Red- Germination is epigeous and the plantlet wood’ given to this species in California) has 2 cotyledons, rarely 3 (Debazac, and is formed of tracheids with bordered 1964).
foundation population (Donnet, 1984) for Sequoia sempervirens grows on podzol- a ic reforestation programme to exploit the in- or clay to silty loam soils (Lindquist, a dustrial value of the species. Its industrial 1974) and on limestone-rich soils. It is gen- value is that: 1) it rarely suffers from dis- erally found between 30 and 750 m above eases or from attacks by insects (Dufre- sea level. It requires high humidity, the noy, 1922; Bull, 1951; Roy, 1966; Gale, Californian climate meeting this require- 1962) (when young its main enemy is fire ment with frequent fog in summer and (Lindquist, 1974)); 2) its vigour and long abundant rains in winter (Lindquist, 1974). life are exceptional: the 2 tallest trees Young sequoia is adversely affected by known in the world (ARC 154 and ARC temperatures < 0°C, but tolerates low light 28*) belong to this species. The age of a intensity (Donnet, 1984). 112-m high and 4.6-m thick tree has been Juvenile growth is very rapid (Donnet, estimated at 2 200 years (Lindquist, 1974). 1984), and is not seasonally rhythmical. In France the storm of 1982, which was of Franclet (unpublished results) has ob- exceptional violence, revealed the ex- served a growth arrest in old trees during tremely good wind-throw resistance of the poor seasons, when the terminal buds be- sequoias (de Champs et al, 1983); 3) its big, round, and covered with green come heart-wood is coloured, light (density 0.40 scales. Fruiting generally starts at≈ 15 yr; to 0.45), easy to work, resistant to bad cli- sometimes, however, fruiting occurs on 2- matic conditions and to pests. Its phenolic yr-old seedlings when ecological condi- products confer exceptional chemical sta- tions are unfavourable (Franclet, unpub- bility (Gale, 1962). It is utilized as timber lished results). for woodwork, industry and horticulture sometimes Sprouts the develop on and also for plywood production (Panshin trunk, and root suckers have been ob- et al, 1964). It has potential value in the served (Libby, personal communication), paper industry, although stump sprouts are the root origin of which has been con- preferred because of the pale colour of the firmed by the authors. The morphology of sapwood and its fiber length, which make the young sprouts and root suckers is typi- this wood a choice raw material for pulp cally juvenile. produced by the modern CTMP (chemical thermo mechanical pulping) technique, re- garded as the pulp of the future by many Value and uses specialists (eg the Technical Centre of the Paper Industry at Grenoble) (personal communication). Sequoia sempervirens is In France, Sequoia sempervirens has his- very well suited for short rotation coppic- torically been considered as an ornamental ing, which would provide the stump sprout tree, and has not been used in silviculture supply required. because of its low germination rate and the to cold of the young trees. The priority now is to select superior susceptibility But in recent years interest has increased trees from those tested over the course of in its use in forestry, because of the many time in the field for their cold resistance, old trees which have survived very well the which is the ecological factor most limiting harsh winter of the French atlantic region. to its planting in Europe. Rapid, large-scale These old trees could provide the basis of propagation of these ’plus trees’ will re- * ARC 28 ARC Arcata forest. ARC 154 No of the tree by the National Geographical = registration or = Institute of the USA.
axillary buds, elongation of shoots and for quire techniques vegetative propaga- rooting of these shoots. Rooting can partly tion. or totally be achieved in vitro. Micropropa- gation is considered to be successful when acclimatization of the plants ex vitro can be Horticultural vegetative propagation achieved reliably. The superior trees (’elite’ trees) identified will be adult, and in many cases very old, Initial studies which makes vegetative propagation diffi- cult. studies were conducted by Restool Early Attempts at vegetative propagation by (1956), who studied the dependance of the cuttings in California (Becking and Belleto, behaviour of shoot segments from burls on 1968; Libby and Mc Cutchan, 1978; Libby, factors such as position of the explant in 1982) as well as in France (Franclet, the stem, weight of the explant, composi- 1981),have shown that for cuttings of old tion of the culture medium, and environ- sequoias, rooting is difficult, later growth is mental conditions. slow and plagiotropic. For the propagation Murashige (1977) later tried to develop of such trees, sprouts or suckers must be culture medium to increase in vitro micro- used (Lindquist, 1974; Franclet, 1981; a propagation, and obtained rooted plants Poissonnier et al, 1981). Such shoots are from sprouts of adult trees. rarely available in great numbers under natural conditions, but certain techniques, Inspired by this work, Boulay (1978) at- such as cutting back of the main trunk or tempted to culture materials taken at differ- root-heating via industrial water (Cormary ent heights from trees of various ages (5, et al, 1980) can increase their production. 20, 50 and 100 yr), and found that only ≈ sprout shoots could be cultured. After a re- These difficulties have stimulated fur- ther research work (Festa and Gambi, iterated culture sequence on multiplication medium (MM) and on elongation medium 1978; de la Goublaye, 1981; Vershoore- (EM), many shoots were obtained which Martouzet, 1985), encouraged by the de- could be rooted ex vitro. monstration that the species can be cul- tured in vitro (Ball, 1950; Restool, 1956). This work provided the foundation for In reviewing the in vitro micropropaga- many later studies of importance of the tion of Sequoia sempervirens, it is neces- physiological state of the material, on the sary to separate micropropagation in sen- effect of reiteration of the subcultures, on rooting conditions and on behavior of the stricto from other regeneration su strategies. cuttings ex vitro. SENSU STRICTO MICROPROPAGATION Sterilization of culture media and disinfection of the material Micropropagation in sensu stricto consists Disinfection techniques for excised materi- of the use of miniaturized explants bearing al must be established by the experimenter either pre-existing caulinary meristems or meristematic areas of leafy axils (Boulay, for each type of explant (Boulay, 1985). One recommendation (de la Goublaye, 1985). It comprises 3 phases: multiplica- tion by intensive and rapid development of 1981; Verschoore-Martouzet, 1985) is to
coat the transversal sections of the ex- Regarding the meristems, Verschoore- cised stem segments with paraffin to pre- Martouzet (1985) found no contamination after using calcium hypochlorite, irrespec- vent penetration of the disinfectant. In gen- tive of the date of removal and origin of the eral, the disinfection protocols adopted explant in an 80-yr-old tree. Walker (1986) have included immersion in a commercial also found none, even without using disin- solution of sodium hypochlorite or a filtrate fection treatment. of a calcium hypochlorite suspension, pre- ceded in some cases by pretreatments with 10% hydrogen peroxide (Ball et al, Multiplication phase 1978; Ball, 1987), or soaking in liquid soap, followed by rapid dipping in 70° GL ethanol (Boulay, 1978), or benlate fungi- Explants used were stem segments (fig cide solution treatment (Bekkaoui and 1 a), bearing 5 to 8 leaves. The MM culture Tranvan, unpublished results). Before in- medium used during the multiplication troduction in vitro, explants have generally phase was derived from that of Murashige but not always (Ball, 1987), been rinsed 3 and Skoog (1962) with auxin and cytokinin times with sterile distilled water. Infection added. The media used by different au- thors have been summarized by Boulay rates are always high in material from adult (1989). In our laboratory, the MM multipli- or old trees (Boulay, 1978; Verschoore- cation medium used contains BAP (2.2 10-6 Martouzet, 1985; Bekkaoui and Tranvan, M) and NAA (5 10 M) (tables I, II). The -8 unpublished results). They vary according period of culture on MM varies from 3 to 8 to the original position of the explant with weeks (Boulay, 1978; de la Goublaye, infections being particularly important on 1981; Fouret, 1987; Tranvan et al, 1991). explants originating from the top of the tree (Ball et al, 1978; Franclet, 1981; de la Gou- After axillary buds had developed (fig blaye, 1981),and with the season of their 1b), newly formed shoots were isolated removal. Infections are less frequent in and transferred to identical fresh medium material explanted in July (de la Goublaye, for intensive multiplication. They can be 1981).For grafted material, the period transferred to an EM elongation medium from March to June is favorable (Vers- (fig 1 c), and the obtained stems (fig 1 d) di- vided for multiplication on MM. choore-Martouzet, 1985).
The material can be grown under weak without activated charcoal et al, (Walker lighting and a wide range of temperatures, 1985). with a preference for temperatures of But more generally, axillary buds which ≈ 24°C. develop during the multiplication phase (fig A multiplication ratio from 3 up to 8 can 1b) are isolated and transferred to the EM be achieved at 3 weekly intervals (Boulay, medium (fig 1 c) based on the Murashige 1985). The ability of the explants to devel- and Skoog (1962) formula, again without op axillary buds depends on the chrono- hormones, but containing activated charcoal logical age of the mother plant and the (tables I, II). The favourable effect of activat- original shoot, and on the original position ed charcoal on growth of Sequoia sempervi- of the material on both the ortet and ramet rens in vitro has been previously reported (Boulay, 1978; de la Goublaye, 1981; by Boulay (1978). The time of culture on EM can vary from 4 to 8 weeks (Boulay, 1978; Verschoore-Martouzet, 1985). Thus, ex- plants originating from a sucker are more Fouret, 1987; Tranvan et al, 1991). reactive than explants derived from the The behaviour and the morphology of crown, and for the same sucker the most the shoots differ over time according to the apical regions (most recently formed) physiological state of the material intro- show the best response. duced into culture. This state depends pri- marily on the original position on the moth- er plant and its chronological age: thus de Elongation phase la Goublaye (1981),comparing the behavi- our of 3 topoclones (top and base of the An isolated cultured meristem will elon- crown, base of the trunk) of clone S5 from gate into a leafy shoot if transferred to a a tree aged at least 500 yr (Franclet, modified Murashige and Skoog (1962) me- 1981),observed that after 1 subculture the dium without added growth regulator, and topoclone from the top of the crown grew
lowest. Later Fouret (1987) noticed that and persisted for a long time. Thus ma- gin the growth of materials cultured in vitro for terial of sucker origin yielded better results several years was distinctly slower in a than the inferior branches of the crown and clone (No AFOCEL 78461 clone II in our better still than the superior branches (de la = laboratory) from a 500-yr-old tree (ARC Goublaye, 1981).Likewise, the morphology 154) than in clones from a 1-yr-old (No of shoots from the old material (clone II) re- AFOCEL 83753 clone J) or 50-yr-old (No mained different to that of shoots from the = AFOCEL 77304 clone I) trees. young material (clone J) for several months = (Fouret, 1987). But after 4 yr of repeated Fouret (1987) reported that, at the end subcultures the clone originating from a of the elongation phase, when the initial shoot of the top of the 80-yr-old NP29 tree material was young, the leaves were long, (Verschoore-Martouzet, 1985) behaved in soft and light in colour. Furthermore, the morphology and reactivity like the clone ob- phyllotaxy was of axial type in a clone from tained from the sucker of the same tree a 1-yr-old tree, whereas it was either of ax- (Franclet, personal communication). ial or crosswise-opposite type in a clone from a 50-yr-old tree, and of crosswise- Verschoore-Martouzet (1985) observed opposite type or more often distichous type that, with NP 29 tree material, shoots de- in a clone from a 500-yr-old tree. veloped from stem segments after 3 or 4 subcultures on medium supplemented with After 2 months on EM medium, stems cytokinin had orthotropic growth, whereas sometimes rooted spontaneously although the shoots which developed initially in vitro without any auxin, but no rooting was ob- plagiogropic. were served when the material originated from the very old tree (Fouret, 1987). Micropropagation using passages through EM (-MM-EM- sequence) avoids After a culture on EM of > 3 months the the abnormalities of waterlogging and fas- stems from clone II sometimes showed ba- ciation, which often appear in protocols in- sal or apically situated outgrowths from volving several subcultures on cytokinin which slender shoots might grow (Bek- containing medium (Fouret, 1987; Boulay, kaoui et al, 1984; Fouret, 1987). 1989) and increases shoot production. If the period of culture on EM lasted for 4 months or longer (Tranvan, unpublished results) the shoots of the old material Rooting and acclimatization phases (clone II) (fig 2a) rooted spontaneously un- less they stopped growing and their apical The shoots obtained in vitro can either be bud was getting round (fig 2b), after which, rooted vitro under horticultural if growth took up again (fig 2c,d), very mor- directly ex conditions, or rooted in vitro prior to accli- phologically different areas were observed matization ex vitro. to have developed along the stem. vitro Rooting ex Effect of repeated subcultures In the first studies, shoots could not be rooted in vitro, but had to be rooted ex vitro Although during repeated subcultures ac- cording to the MM-EM sequence the ma- (Boulay, 1978). Later, Poissonnier et al (1981) proposed the use of cold storage of terial became increasingly reactive (Bou- lay, 1985), differences in reactivity the in vitro shoots and use of specific sub- correlated with the position of explant ori- strates to improve rooting.
The improvement of shoot production which various dilutions of modified were and the improvement of the ex vitro rooting Murashige and Skoog (1962) medium with after repeated subcultures enabled > an addition of auxin. Using materials from 200 sprouts of adult trees, Ball et al (1978) ob- clones originated from adult trees selected tained 20% of rooted shoots with in various regions of France and California an or- = thotropic growth habit after acclimatization, to be multiplied (Franclet et al, 1987). although it has to be emphasized that the These cuttings were used for the establish- sprouts used were physiologically young ment of a mother tree orchard at Guingamp material (see Rejuvenation). in Brittany, from which orthotropic shoots could be recovered in the longer term by In an attempt to optimize in vitro rooting, pruning and cutting back operations for Bekkaoui et al (1984) studied the behavior commercial production of cuttings. of material from 2 clones (I and II) obtained from a 50 and 500 year-old tree respec- After observing effects on conventional tively, using multiplication and elongation propagation of the original location of the techniques similar to those of Boulay cuttings on adult trees, de la Goublaye (1978). The apical microcuttings removed (1981), using an 80-yr-old tree located at after the elongation phase were 1 to 2 cm Fontainebleau, studied the effects of to- long and had 8 leaves. Optimal conditions shoot growth habit in vitro pophysis on for rooting were found to be: rooting induc- during repeated subcultures according to tion (1 wk) on a RIM medium supplement- the MM-EM sequence. She found there ed with auxin (NAA 5 10 M); rooting ex- -5 was a progressive improvement in rooting pression (6 wk) on root expression (REM) speed (ex vitro) and, more slowly, in the medium, identical but without auxin (tables recovery of orthotropy. However, the topo- I, II) (temperature: +20 to 25°C); daily illu- physical effects did not totally disappear mination: 9 h (100 W m for clone I; 9 h ) -2 over the period of the subcultures. With (100 W m + 15 h red (12 to 15 W m ) -2 ) -2 one of the topoclones of the Fontainebleau for clone II). There was a very distinct dif- tree the number of subcultures not only af- ference in reactivity between the 2 clones, fected recovery of orthotropy but also later with the percentage of rooted explants and wood productivity ex vitro (Franclet, un- the mean number of roots per explant al- published results). A similar result was ob- ways inferior in the oldest material. The tained in an experiment conducted on ma- best results obtained were in clone I, 90% 500-yr-old terials from a tree, originating rooting, with a mean of 6 roots per explant; from California. in clone II, 60% rooting and mean of 3 roots But despite this progress, the problems per explant. The material from the 500-yr- of long rooting initiation period, rooting fluc- old tree was also more limited in its ability to tuations according to season and plant ma- adapt to the variety of in vitro culture condi- terial used, persistant plagiotropic growth tions used. Rooting ability and maintenance remained. Consequently studies were un- of this property under widely varying envi- dertaken on the effects of in vitro rooting. ronmental conditions appeared to charac- terize juvenility. in vitro and acclimatization Rooting In further work Walker et al rooting a on (1985, 1987) applied the following treat- In initial with materials from experiments ments to microcuttings from clones ob- sprouts of different aged trees, Boulay tained from young and old trees: a root in- (1978) obtained an in vitro rooting rate of 5 duction phase under dark conditions (5 d); to 25% depending on the media used, an expression phase (35 d) under different
the recovery by old plant material of at of lighting; an acclimatization phase types as least some of the properties of younger ma- under natural light, with additional lighting terial (see Pierik, 1990). Walker (1985) pre- to give a 16 h photoperiod. For the young fered the expression ’rejuvenilization’, re- easily rooting material, acclimatization was serving the expression ’rejuvenation’ for the accelerated when rooting was obtained rapid and total recovery of juvenile charac- under a high quantum flux (up to 280 μE ter, for example in apomictic or zygotic em- ). -1 ·s -2 m The old material was difficult-to- bryo formation (Franclet and Boulay, 1989). root, but a high quantum flux increased the rooting rate and number of roots. Under In situ pruning, cutting back or grafting short days, night breaks of red or far red can induce a rejuvenating process (Ver- light had no effect. For the older clone ac- shoore-Martouzet, 1985), improving in vitro climatization also depended on the period performance (Franclet, 1981; Franclet et the rooted shoots were cultured on agar al, 1987). The effect of these techniques is medium. After 10 d they could be acclima- to bring the root system closer to the tized with 70% success rate. above ground shoot system (Doorenbos, 1965; Chaperon, 1979; Franclet et al, The practice in laboratory (Fouret, our 1980; Favre, 1980). The role of roots could 1987; Fouret et al, 1989; Tranvan et al, be to supply cytokinins (Kende, 1964; Itai 1991) has been to take apical cuttings 6 Vaadia, 1965; Sitton et al, 1967). and in length from material maintained in vi- cm tro for rooting induction in short days after Media containing a cytokinin appear to culture on EM (induction: 1 wk; expression: have a rejuvenation effect (Boulay, 1978; 6 wk) (fig 3). If the culture period on EM de la Goublaye, 1981).For example, sub- exceeds 3 months, the apical cuttings pro- cultures on a medium supplemented with gressively lose their ability to root cytokinin increases the K/Ca ratio in mat- (’experimental aging’; Tranvan: unpub- erials from an 80-yr-old tree to values char- lished observations). For clone II the num- acteristic of seedlings. Similarly, the peak ber of subcultures has affected in vitro ’peroxidase activity/total proteins’ increas- rooting (Arnaud et al, 1987). At present, af- es with the number of subcultures on cy- ter 12 yr of repeated in vitro culture, shoots tokinin-containing medium (Verschoore- from clone II often root spontaneously. Martouzet, 1985; Boulay, 1987b). These ratios are correlated with rooting ability. These results showed that it was logical to REJUVENATION consider the in vitro culture as a technique utilizable for attempting to rejuvenate plant material. In addition, this was suggested Different kinds of aging occur in trees (For- by Franclet (1981), Nozeran et al (1982), tanier and Jonkers, 1976; Chaperon, Margara (1982) for various materials. 1979). Chronological age is the duration of with Several protocols developed were time since germination. Physiological ag- double aim: a ing reflected, for example, in rooting ability, is the result of the increase in tree size and to study the conditions and mechanisms - complexity. Ontogenetic age reflects the involved in the rejuvenation process (basic successive phases in development, re- aim); vealed for example in topophysis (Seeling- attempt the recovery of all ’juvenility to - 1924; Franclet, 1983; Boulay, 1987b). er, the orthotropy in properties’, particularly very old trees material issued from adult Rejuvenation is a necessary prerequisite or mass propagation, and can be defined for (applied aim).
Single or repeated cultures of isolated very old tree has thus not yet been caulinary meristems (protocol IM) achieved. Meristems (0.1 mm shoot tips, including Micrografting of apices the meristematic dome with 1 or 2 leaf pri- from old material mordia) were removed (Verschoore- onto young rootstocks (protocol MG) Martouzet, 1985) from an 80-yr-old tree at different periods of the year from shoots The effects have been studied of grafting situated at the base of the tree or at 15 m aged axillary branches apices from unroot- height from suckers, or from the same or- ed shoots of clone II onto vitroplants of the gans grafted onto seedlings, and from clone J (Tranvan et al, 1991).The shoots seedlings from the same tree. The shoots providing the apices were maintained on developed from these meristems were the EM medium (elongation medium) of found to recover some juvenile characters the MM-EM sequence for 2, 4 or 6 after culture on a modified Murashige and months. The apex (= 4 mm) was grafted Skoog medium with IAA (Boulay, 1989): eg onto an oblique section of the rootstock at typical juvenile morphology, good rooting 1 cm distance from the roots (fig 4a). ability, partial recovery of the orthotropy. The time of year of removal had little influ- When the grafted apices grew, the ence, but ’memory’ of topophysis was not scions acquired a growth rate and mor- phology similar to those of clone J (fig 4b). eradicated. Shoots were then excised and submitted With repeated subcultures of meristems inducing to root treatment to according a issued from a very old tree (ARC 154: 500 Bekkaoui et al (1984). Micrografting ap- yr), the shoots progressively recovered a peared to increase the number of shoots young physiological state, evaluated by a which rooted, speed of rooting and quality better rooting ability (realized ex vitro), by of the root system (fig 4c). After transplant- the acquisition of the ability for adventitious ing ex vitro the rooted stems had an ortho- budding on isolated leaves and by a better tropic growth habit for several weeks, but reactivity* of the isolated apical meristems then reverted to plagiotropy. However, a (Walker, 1986). But the plants still main- very small number of plants were still or- tained plagiotropic growth in the green- thotropic 2 yr later (fig 5). house. Total (according to the praticians) or ontogenetic (see Schaffalitzky de Muck- Thus micrografting initiated a rejuvena- adell, 1959) rejuvenation of material from a tion process. This technique is currently * days necessary ’to get the number of the cultured meristem green The ’meristem test’ is based on again’.
protocols were tested, differing in frequen- being improved in our laboratory (Tranvan, cy of subculturing on 2 different media and unpublished results). The efficiency of this technique was confirmed by Huang et al in sequence of using these media. Efficien- (1992) who studied morphological and pro- cy of rejuvenation of each protocol was tein markers of rejuvenation status. evaluated for 18 wk after the start of the culture considering the modifications in a number of properties (Fouret et al, 1985; Repeated subculture Franclet et al, 1987): improvement of of stem segments (protocol RS) growth activity of the longest axillary stem, reappearance of a young type phyllotaxy, reduction of the reactivation time of the iso- (1987) and Fouret et al Fouret (1985, lated caulinary meristems, increase in the 1986a, 1989) used stem segments with 3 rate of rooted explants and the reduction of leaves (thus 3 axillary meristematic areas) rooting time (Fouret et al, 1985), increase from different-aged trees (1 yr (clone J), in mean root number per explant, improve- 50 yr (clone I) and 500 yr (clone II)) to ment of the quality of the root system. In study the effect of repeated subcultures. addition, we observed that the increase of The explants were cultured on MM medi- um containing BAP (2.2 10 M) and NAA -6 the endogenous IAA/ABA ratio was corre- (5 10 M) or on MO (same medium as -8 lated with rooting ability of the shoots (Fou- MM but without growth regulator). Eight ret et al, 1986b; Fouret, 1987) and another
conditions during the acclimatization pro- criterion was the ability to produce adventi- cess which can greatly influence root sys- tious buds on isolated leaves (Fouret, tem quality. How such rejuvenated materi- 1987). The total or ontogenetic rejuvena- al will grow in the long term is uncertain. tion is considered as successful when the One of the first trees produced by epiphyl- vitroplants exhibit continuous orthotropic lous budding (during the IM protocol of growth after ex vitro transfer (Fouret et al, Walker, 1986) is 5.2 m tall after 5 seasons 1989). compared to 0.70 m for the control ob- Rejuvenation was more difficult to ob- tained using the technique of Boulay tain in clone II than in clone I. The results (1978) (Franclet, unpublished results). showed the prime importance of the ’fre- These results indicate that several fac- quences of the subcultures’ for the rejuve- tors control the rejuvenation process: the nation of old material. Frequence of ’one release of shoot meristems from the correl- subculture per week’ was most efficient, ative control, the repeated subcultures on but it was also beneficial to apply a rejuve- medium supplemented with cytokinin, and nation-inducing treatment (3 transfers on the closeness of the shoot meristem to the MM for example), and to conduct the other root system. transfers on MO. In this work, the juvenile properties ap- pear successively more or less rapidly and IN VITRO REGENERATION more or less clearly according to the proto- col and the physiological state of the initial Horry-Charrier (1988) isolated protoplasts material. But certain properties of the reju- from foliar tissues of vitroplants in young venated state may disappear (Arnaud et material and produced microcalli of 30 al, 1989). The rejuvenation - ’rejuveniliza- = cells, but without regeneration. Regenera- tion’ according to Walker (1986) - appears tion has, however, been obtained by other as a sequence of ability states. The methods. cuttings from the material rejuvenated ac- cording to the RS protocol kept a plagio- tropic growth habit ex vitro. Formation of somatic embryos Adventitious budding induced on isolat- ed leaves of previously rejuvenated materi- al in one of the protocols produced plants Bourgkard and Favre (1988, 1989) ob- (Fouret et al, 1989) which maintained ex tained somatic embryos in calli on mature vitro orthotropic growth for 4 yr in the zygotic embryos and on cotyledons and greenhouse and then outdoors. It has from isolated hypocotyl segments been recently observed (Monteuuis-Bon, 7 d after germination. This vitroseedlings unpublished results) that the clonal materi- embryogenesis was only observed when al produced by these epiphyllous buds seeds recently collected or stored in a cold contained a specific protein profile. Onto- 11 to 12 months (darkness; room for < genetic rejuvenation has thus been ob- +4°C). Embryogenic potential in regularly subcultured calli was maintained for only tained in clone II from a 500-yr-old tree (ARC 154) with this specific protocol. How- 12 wk. In this embryogenesis, no proem- bryo was observed, and the somatic em- ever, in such experiments it is difficult to bryos with typical bipolarous structure distinguish the contribution to shoot growth were obtained in darkness on culture medi- modalities of material rejuvenation, root um containing KIN 2 μM, BAP 2 μM and system morphology and environmental
2.4-D 2.5 μM, without a sequence of me- extruded from the bottom of the gameto- dia of different composition. The callus ob- phyte cavity, where the embryo used to be, tained was solid and consistent in charac- with some buds already present. After 6 months’ culture (with transfers at monthly in- ter, and the somatic embryos were mixed with adventitious buds. On a medium with- tervals) the callus was covered with shoots, which could be removed and grown on oth- out hormone and under light, the somatic embryo shoots grew, but most of their roots er media into orthotropic rooted plants. turned brown and developed lateral roots. Many of them have subsequently devel- oped into small trees (Ball, 1987). The chro- In our laboratory (Lefrançois, 1988), mosome number of the female gameto- neoformed buds (fig 6) (cf Formation of phyte derived plants was 66. Chromosome adventitious buds) were isolated from calli counts of living female gametophytes of from mature zygotic embryos and placed seeds have given various counts; it is as- on elongation medium. These buds devel- sumed that the occasional diploid cells di- oped into leafy stems (fig 7), and, from the vide to produce the callus from which the 12-cm stems, the ten 1- to 3-cm long ≈ buds regenerated (Ball, 1987). subapical leaves were removed and put through a double treatment consisting of caulogenetic then callogenetic media Mature zygotic embryos and (Krogstrup, 1986; Lelu, 1987). On one isolated cotyledons of these embryos leaf, a soft translucent white callus was formed which contained very numerous similar to that of Lelu et al Using protocol a polarized structures (fig 8). Each structure (1987) with mature embryos and isolated with 2 parts, one with a meristematic cell cotyledons, organogenous calli have been cluster and the other with long vacuolized found to develop with caulinary neoforma- cells, is similar in appearance to young zy- tions, and in a lower amount with root neo- gotic embryos (Singh, 1978). These struc- formations (Chiffaudel and Stroobants, tures were also similar to the somatic pro- 1987). Vascular connections pointed be- embryos observed in other gymnosperms tween the caulinary neoformations and the (Gupta and Durzan, 1986; Hakman and callus tissues. According to Haccius Fowke, 1987; Lelu et al, 1987). (1978), these buds were adventitious buds and not somatic embryos whose radicular pole would have aborted. After transfer to Formation of adventitious buds elongation medium, these buds developed leafy stems, rooting either spontaneously or after induction (RIM-REM treatment). Female gametophytes More recently, Tranvan (unpublished re- sults) has observed that adventitious buds Plantlets have been regenerated from cul- can develop on isolated cotyledons without tured female gametophytes of Sequoia a callus stage (fig 9). sempervirens (Ball, 1981, 1987). Mature seeds were excised from green cones Bourgkard and Favre (1988, 1989) also which were not yet opened. The cones and obtained compact organogenic callus on then each individual seed were surface- mature entire zygotic embryos or divided sterilized by soaking in full-strength bleach young seedlings in which adventitious (Ball, 1987). After removal of testae, the buds were mixed with somatic embryos, thin layer of female gametophytes (naturally and observed that caulogenesis de- axenic) was isolated from the embryo. After creased, as did embryogenesis, with the 2 months on the culture medium, a callus duration of seed storage at + 4°C. =
Stem segments greenhouse. By contrast, plagiotropic stem segments from the crown formed few ad- ventitious buds, and these buds only grew Ball (1950) obtained callus capable of re- slowly and never produced acclimatized generating buds from stem segments is- plants. sued from burls (this was the first in vitro culture in Sequoia sempervirens) and in Ball et al (1978) and Ball (1987) ob- 1978 Ball et al obtained numerous adventi- served that Wolter and Skoog’s mineral tious buds from juvenile and orthotropic mixture was better than Murashige and stem segments from the base of an adult Skoog’s medium for regeneration from tree. These buds developed from cortical stem explants (growth regulators were proliferating tissues through ruptures in the 2.4.5-T and SD 8339). After elongation on a medium lacking cytokinin and containing epidermis into orthotropic shoots which activated charcoal and auxin, the shoots could be rooted and transplanted in the
type SR protocol (Fouret transferred into rooting mediumI to the et cording were al, 1989), rooted easily in vitro after induc- (with IBA) for 12 h, and then to rooting me- tion and acclimatized easily. They had the dium II (without auxin and with activated morphology of young plants, were vigorous charcoal). After 30 d, shoots were trans- and showed orthotropic growth (fig 11) (cf ferred to a mixture (2:1:1) of sphagnum Repeated subculture of stem segments moss perlite vermiculite in tubes irrigated (protocol RS). By contrast, the non- with a mineral solution. The tubes were rejuvenated material of the same clone re- stored in a greenhouse. Ball (1987) ad- mained plagiotropic (fig 12), and excep- vised that a large root system should be tionally produced male cones on some established before plantlets were trans- plants only 21 months after transplanting in ferred to soil. the greenhouse (fig 13) (Tranvan, unpub- lished results). Leaves The adventitious budding technique has great potential for multiplication, but the Ball et al (1978) and Ball (1987) observed buds obtained after a callusing stage may that isolated leaves, cut transversely in not be true-to-type genetic, as Patel and half, and laid on culture medium supple- Berlyn (1982) have observed for Pinus mented with cytokinin SD 8339 and auxin coulteri. However, Ball (1987) counted 2.4-D, required an intervening callus stage chromosomes in root-tips of Sequoia sem- produced on the section surface for regen- pervirens trees regenerated from stem eration of buds. Nevertheless, adventitious pieces and leaf inocula and found that in buds have been produced without appar- both cases the plants are regenerated only ent proliferating callus by: diploid cells. from in vitro basal leaves from maintaining - material (clone J) or, in clones J,I young and even II, on basal leaves of the stem in RECOMMENDED PROTOCOL contact with the culture medium during RS FOR THE MICROPROPAGATION OF SEQUOIA SEMPERVIRENS rejuvenation protocols (Fouret, 1987); after specific treatment of subapical - (young) isolated leaves from clone II Regeneration protocols have been detailed during the rejuvenation protocols IM, Walk- by Ball (1987). Described below are our er, 1986), RS (Fouret, 1987; Fouret et al, recommended variants to the protocol pro- 1989) or MG (Tranvan, unpublished posed by Boulay (1989). results) (fig 10). The epiphyllous bud inducing treatments consisted of 2 stages: Culture establishment induction (3 wk) on BM medium (similar to MM but without NAA and with BAP 1 mg· ) -1 l and then expression (4 wk) on the Choice of initial explant BMO medium (similar to BM but without BAP). Stem pieces should be taken from basal Sometimes elongation of such buds is sprouts of an old tree or from actively difficult to achieve (Tranvan, unpublished growing grafted plants grown in the green- results). Nevertheless the leafy shoots, house. If the explants are taken from the which grew from epiphyllous buds induced top of an adult or old tree, July is the best on material of clone II and rejuvenated ac- period to initiate sterile culture.
upon the season, environmental conditions Disinfection and sterilization of the mother plant, age, situation on the tree and lignification status of the explant. The stem segments should be soaked in benlate solution (≈ 0.2%, about 24 h) or in liquid soap (2 or 3 min), rinsed in water Choice of miniaturized explants and then ethanol at 70° GL (1 or 2 min), soaked in a commercial sodium hypochlor- It is recommended to small, little use or ite solution (5 to 20 min), and finally rinsed lignified stem segments (≈ 15 mm in non 3 times with sterile distilled water. height with some leaves); or preferably caulinary apices (meristem surrounded by The duration of these treatments and the some foliar primordia). concentration of the solutions used depend
Rooting induction is achieved by culture on Multiplication one-third strength Murashige and Skoog medium as modified by Boulay (1978), Stem segments or meristems are placed with NAA (9.3 mg·l (RIM) for 1 wk. Root- ) -1 in or on multiplication medium (MM) which ing expression is achieved on a similar me- is composed of half-strength Murashige dium without auxin (REM). and Skoog medium as modified by Boulay then be Two methods applied: can (1978), NAA (0.01mg·l BAP (0.5 mg· ), -1 ), -1 l sucrose (30 g·l and agar (7 g·l ). -1 ) -1 Method A: Approximately 30 d after the ap- pearance of the first roots, the vitroplants are transferred vitro to a perlite/ ex vermiculite mixture watered with a mineral Elongation nutrient solution (for example, Phytotron solution) (de Bilderling and Lourtioux, After 1 or 2 months, the axillary buds (or 1976) or AFOCEL solution (Poissonnier et the very short axillary stems) should be al, 1981) and then transferred to a horticul- transferred to an elongation medium (EM), tural substrate. similar to MM but without growth regulator Method B: Approximately 10 d after the ap- and containing activated charcoal (20 g l ). -1 pearance of the first roots, the vitroplants Micropropagation is achieved by using are maintained at cool temperature (eg sections (5 or 6 leaves) of vitroshoots, el- 22°/12°C) under natural light for 1 wk; they ongated on EM for 1 or 2 months, for cul- are then transferred ex vitro, enclosed in a ture on MM (several MM-EM cycles). Melfert ’envelope’ and placed on a thick moist cloth maintained at 22°C by bottom heat. When roots are visible in the contain- Rooting and acclimatization (weaning) er, the plants are transferred to a horticul- tural substrate. Ex vitro rooting and weaning In both cases, the plastic tent must be gradually removed in order to progressive- It is recommended to harden the vitro- ly reduce air moisture. shoots for 1 or 2 wk at a temperature of 5 to 10°C under natural light (10-13 h illumi- nation). The cuttings should then be Rejuvenation soaked for 24 h in an auxin solution and = transferred to a heated rooting substrate If rooting of the material is difficult, mother and maintained under a mist system or en- plant material should be submitted to a re- closed in apropagator (photoperiod: 16 h). juvenation protocols prior to root induction After 6 to 8 wk, the cuttings should be treatment. Three kinds of in vitro rejuvena- transferred to Melfert ’envelope’ and the tion protocols can be used: 1) single or re- aerial parts acclimatized by progressive re- peated culture(s) of isolated caulinary mer- duction of relative humidity. istems; 2) single or repeated micrografting (s) of apices from old material onto young rootstocks; 3) repeated subcultures of In vitro rooting, transfer vitro ex stem segments. and acclimatization If the vitroplants remain plagiotropic af- Terminal vitrocuttings≈ 6cm high should ter transfer ex vitro it is recommended to be taken from shoots elongated on EM. submit the mother plant material to a reju-
venation In the USA the Compa- Co) in France. protocol leading to epiphyllous taux budding. The shoots produced from the nies Simpson Timber and Georgia Pacific epiphyllous buds can then be induced to produce vitroplants (Boulay, 1987a) follow- root, or introduced onto the MM-EM cycle, ing a scheme initiated by Ball et al (1978) before rooting induction. adopting the technique of Boulay or (1978). PRACTICAL APPLICATIONS PROSPECTS These techniques have been in continuous practice for≈ 14 yr, some with clones from The interest of Sequoia sempervirens is recognized in the forestry and paper indus- very old trees (for example ARC 154, ARC try. But future prospects for large-scale mi- 28). cropropagation for reforestation purposes Boulay (1987a) has reported the AFO- will depend upon economic considerations CEL micropropagation techniques for the (the present methods are still too expen- production of rooted clones from 120 se- sive for mass production) and increased lected adult genotypes with a view to refo- basic knowledge of rejuvenation process- restation. es. Concerning this last point, the Sequoia The technique of Boulay (1978), later sempervirens is considered as good exper- modified in our laboratory, involving alter- imental material for studying rejuvenation nate subcultures of 4 to 8 weeks on a = in woody species (Fouret et al, 1986a; medium containing an auxin and a cytoki- Boulay, 1987b; Franclet et al, 1987). The nin, followed by a medium lacking growth search for specific gene expression asso- regulator but containing activated charcoal, ciated with rejuvenation has already started allows the vitrostems of Sequoia to be (Bon-Monteuuis, unpublished results; maintained and to multiply for period of Huang et al, 1992). Moreover, the control of years without the ’clonal degeneration’ somatic embryogenesis, associated or not found in Douglas fir (Bekkaoui et al, 1986). with transformation experiments, constitutes By using this technique the rapid multi- an extremely interesting task for the future. plication of > 400 clones of old trees se- Sequoia sempervirens can be considered lected by AFOCEL scientists in parks and as a good model for the application of these gardens of Western Europe (France, Eng- new biotechnologies to woody species with land, Ireland, Germany and Belgium) has the aim of improving the production of ge- been effected. It has also facilitated the in- netically selected or modified trees. troduction to Europe of almost all 200 clones of the ’Kuser collection’ (a complete sampling of the ’genetic pool’ of Sequoia ACKNOWLEDGMENT sempervirens in its natural area). It has en- sured the multiplication of the conservation We would like to thank Pr E Migniniac for his collections and accelerated the establish- suggestions during useful talks. ment of planting for clonal evaluation at nu- merous sites in France. REFERENCES Some historical or ornamental trees have been commercially propagated using micropropagation eg by RMCV (Re- Arnaud Y, Al Maarri K, Fouret Y, Larrieu C cherche, Multiplication, Contrôle des Végé- (1987) Les régulateurs de croissance exo-
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