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Micropropagation and rejuvenation of Sequoiasempervirens (Lamb) Endl: a review

Y Arnaud, A Franclet, H Tranvan, M Jacques

To cite this version:Y Arnaud, A Franclet, H Tranvan, M Jacques. Micropropagation and rejuvenation of Sequoia sem-pervirens (Lamb) Endl: a review. Annales des sciences forestières, INRA/EDP Sciences, 1993, 50 (3),pp.273-295. �hal-00882846�

Review article

Micropropagation and rejuvenationof 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

(Received 5 May 1992; accepted 17 December 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 appropriatehormonal 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 theseprotocols 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 :revue. Cet article présente les principales caractéristiques botaniques, biologiques et forestières duSequoia 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 lacontourner. 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’obtentionde l’embryogenèse somatique chez cet arbre sont décrites (fig 8). Finalement, l’accroissement denos connaissances en micropropagation in vitro apparaît essentiel pour augmenter la qualité de lasélection clonale et offrir des débouchés pratiques aux travaux concernant l’hybridation somatique etl’embryogenèse somatique. L’acquisition de telles connaissances de base devrait permettre unemeilleure utilisation de cet arbre.

embryogenèse somatique / micropropagation / rajeunissement / Sequoia sempervirens

INTRODUCTION

Sequoia sempervirens (Lamb) Endl is dis-tinguished not only by its exceptional vig-our and long life, but also by its productivi-ty of quality wood. It is a gymnospermbelonging to the Coniferophytes, order Cu-pressales, family Taxodiaceae (Stokey,1981). It is the only species of the genusSequoia (Chadefaud and Emberger,1960). The number of chromosomes is2n = 66 (Stebbins, 1948), with 1 to 6 ac-

cessory chromosomes, according to the

population (Libby and McCutchan, 1978).Several cultivars have been registered andcommercialized (Redher, 1958; Chaudun,1977).

Origin and geographical area

The current genus Sequoia is said to de-rive from an ancestral complex, a compo-nent of which is the genus Rhombostrobus

(upper Cretaceous) (LaPasha and Miller,1981). Sequoia langsdorfii, a fossil speciespresenting narrow parental links with thecurrent Sequoia sempervirens, is sup-posed to have disappeared from Europe atthe end of the tertiary era (Emberger,1968).

The natural area of extant Sequoia sem-pervirens extends from southwest Oregonto California (USA). In the 19th centurysome specimens were exported to Russia,Great Britain and France (Donnet, 1984).

Morphology and development

Sequoia sempervirens is an evergreenspecies, with a thick (up to 25 cm), fibrous,deeply furrowed bark. The heart-wood isreddish brown (hence the name ’Red-wood’ given to this species in California)and is formed of tracheids with bordered

pits, frequently biserial or triserial (Jacqui-ot, 1955). Lacking resinous vessels, theheart-wood is particularly fire-resistantwhen mature. The long fibers (= 4 mm) inthe heart-wood represent 70 to 90% of itsdry weight (Donnet, 1984). The sapwood iswhite and homogeneous (Rol, 1981).

Sequoia sempervirens is a heteroplasticspecies. In very young trees, the long axis(long twig) generally bears acicular leaveswith an axial phyllotaxy. The short axes(lateral twigs) bear light-green, wide andsoft leaves, attached at a right, or nearlyright angle on the axis, with crosswise-

opposite phyllotaxy.In adult or old trees the long, soft, light-

green leaves of the stump sprouts andsuckers are similar to those issued from

seedlings. In the lower part of the crown,the long and short twigs resemble those ofyoung trees. In the upper part of the

crown, the long twigs have squamiformleaves which, in very old trees, look likethose of Sequoiadendron; the short twigsare feather-like, with the leathery awl-

shaped, dark green (sometimes bluish

green leaves), which form an almost acuteangle with the axis of the twig.

In the northern hemisphere Redwoodblooms between November and earlyMarch (Boulay, 1989). The monosporan-giate inflorescences are in a terminal posi-tion on the short twigs (Debazac, 1964).Male cones (< 10 mm long) can also be inaxillary positions and possess numerousstamens. The female cones are 20 to25 mm long, with 15 to 20 woody scales.The seeds weigh on average 4 mg and arebrown, elliptical and bordered by a smallwing. Germination rate is very variable.

Many seeds are often empty, the embryosbadly formed or infected by parasites. Vi-able seeds are stored with difficulty (Don-net, 1984; Bourgkard and Favre, 1989).Germination is epigeous and the plantlethas 2 cotyledons, rarely 3 (Debazac,1964).

Sequoia sempervirens grows on podzol-ic or clay to silty loam soils (Lindquist,1974) and on limestone-rich soils. It is gen-erally found between 30 and 750 m abovesea level. It requires high humidity, theCalifornian climate meeting this require-ment with frequent fog in summer andabundant rains in winter (Lindquist, 1974).Young sequoia is adversely affected bytemperatures < 0°C, but tolerates low lightintensity (Donnet, 1984).

Juvenile growth is very rapid (Donnet,1984), and is not seasonally rhythmical.Franclet (unpublished results) has ob-served a growth arrest in old trees duringpoor seasons, when the terminal buds be-come big, round, and covered with greenscales. Fruiting generally starts at &ap; 15 yr;sometimes, however, fruiting occurs on 2-yr-old seedlings when ecological condi-tions are unfavourable (Franclet, unpub-lished results).

Sprouts sometimes develop on the

trunk, and root suckers have been ob-served (Libby, personal communication),the root origin of which has been con-firmed by the authors. The morphology ofthe young sprouts and root suckers is typi-cally juvenile.

Value and uses

In France, Sequoia sempervirens has his-torically been considered as an ornamentaltree, and has not been used in silviculturebecause of its low germination rate and thesusceptibility to cold of the young trees.But in recent years interest has increasedin its use in forestry, because of the manyold trees which have survived very well the

harsh winter of the French atlantic region.These old trees could provide the basis of

a foundation population (Donnet, 1984) fora reforestation programme to exploit the in-dustrial value of the species. Its industrialvalue is that: 1) it rarely suffers from dis-eases or from attacks by insects (Dufre-noy, 1922; Bull, 1951; Roy, 1966; Gale,1962) (when young its main enemy is fire

(Lindquist, 1974)); 2) its vigour and longlife are exceptional: the 2 tallest trees

known in the world (ARC 154 and ARC28*) belong to this species. The age of a112-m high and 4.6-m thick tree has beenestimated at 2 200 years (Lindquist, 1974).In France the storm of 1982, which was ofexceptional violence, revealed the ex-

tremely good wind-throw resistance of thesequoias (de Champs et al, 1983); 3) itsheart-wood is coloured, light (density 0.40to 0.45), easy to work, resistant to bad cli-matic conditions and to pests. Its phenolicproducts confer exceptional chemical sta-bility (Gale, 1962). It is utilized as timberfor woodwork, industry and horticultureand also for plywood production (Panshinet al, 1964). It has potential value in the

paper industry, although stump sprouts arepreferred because of the pale colour of thesapwood and its fiber length, which makethis wood a choice raw material for pulpproduced by the modern CTMP (chemicalthermo mechanical pulping) technique, re-garded as the pulp of the future by manyspecialists (eg the Technical Centre of thePaper Industry at Grenoble) (personalcommunication). Sequoia sempervirens isvery well suited for short rotation coppic-ing, which would provide the stump sproutsupply required.

The priority now is to select superiortrees from those tested over the course oftime in the field for their cold resistance,which is the ecological factor most limitingto its planting in Europe. Rapid, large-scalepropagation of these ’plus trees’ will re-

* ARC = Arcata forest. ARC 154 or ARC 28 = registration No of the tree by the National GeographicalInstitute of the USA.

quire techniques for vegetative propaga-tion.

Horticultural vegetative propagation

The superior trees (’elite’ trees) identifiedwill be adult, and in many cases very old,which makes vegetative propagation diffi-cult.

Attempts at vegetative propagation bycuttings in California (Becking and Belleto,1968; Libby and Mc Cutchan, 1978; Libby,1982) as well as in France (Franclet,1981), have shown that for cuttings of oldsequoias, rooting is difficult, later growth isslow and plagiotropic. For the propagationof such trees, sprouts or suckers must beused (Lindquist, 1974; Franclet, 1981;Poissonnier et al, 1981). Such shoots arerarely available in great numbers undernatural conditions, but certain techniques,such as cutting back of the main trunk orroot-heating via industrial water (Cormaryet al, 1980) can increase their production.

These difficulties have stimulated fur-

ther research work (Festa and Gambi,1978; de la Goublaye, 1981; Vershoore-Martouzet, 1985), encouraged by the de-monstration that the species can be cul-tured in vitro (Ball, 1950; Restool, 1956).

In reviewing the in vitro micropropaga-tion of Sequoia sempervirens, it is neces-

sary to separate micropropagation in sen-su stricto from other regenerationstrategies.

SENSU STRICTO MICROPROPAGATION

Micropropagation in sensu stricto consistsof the use of miniaturized explants bearingeither pre-existing caulinary meristems ormeristematic areas of leafy axils (Boulay,1985). It comprises 3 phases: multiplica-tion by intensive and rapid development of

axillary buds, elongation of shoots and

rooting of these shoots. Rooting can partlyor totally be achieved in vitro. Micropropa-gation is considered to be successful whenacclimatization of the plants ex vitro can beachieved reliably.

Initial studies

Early studies were conducted by Restool(1956), who studied the dependance of thebehaviour of shoot segments from burls onfactors such as position of the explant in

the stem, weight of the explant, composi-tion of the culture medium, and environ-mental conditions.

Murashige (1977) later tried to developa culture medium to increase in vitro micro-

propagation, and obtained rooted plantsfrom sprouts of adult trees.

Inspired by this work, Boulay (1978) at-tempted to culture materials taken at differ-ent heights from trees of various ages (5,20, 50 and &ap; 100 yr), and found that onlysprout shoots could be cultured. After a re-iterated culture sequence on multiplicationmedium (MM) and on elongation medium(EM), many shoots were obtained whichcould be rooted ex vitro.

This work provided the foundation for

many later studies of importance of the

physiological state of the material, on theeffect of reiteration of the subcultures, onrooting conditions and on behavior of thecuttings ex vitro.

Sterilization of culture mediaand disinfection of the material

Disinfection techniques for excised materi-al must be established by the experimenterfor each type of explant (Boulay, 1985).One recommendation (de la Goublaye,1981; Verschoore-Martouzet, 1985) is to

coat the transversal sections of the ex-

cised stem segments with paraffin to pre-vent penetration of the disinfectant. In gen-eral, the disinfection protocols adoptedhave included immersion in a commercialsolution of sodium hypochlorite or a filtrateof a calcium hypochlorite suspension, pre-ceded in some cases by pretreatmentswith 10% hydrogen peroxide (Ball et al,1978; Ball, 1987), or soaking in liquidsoap, followed by rapid dipping in 70° GLethanol (Boulay, 1978), or benlate fungi-cide solution treatment (Bekkaoui and

Tranvan, unpublished results). Before in-

troduction in vitro, explants have generallybut not always (Ball, 1987), been rinsed 3times with sterile distilled water. Infection

rates are always high in material from adultor old trees (Boulay, 1978; Verschoore-Martouzet, 1985; Bekkaoui and Tranvan,unpublished results). They vary accordingto the original position of the explant withinfections being particularly important onexplants originating from the top of the tree(Ball et al, 1978; Franclet, 1981; de la Gou-blaye, 1981), and with the season of theirremoval. Infections are less frequent inmaterial explanted in July (de la Goublaye,1981). For grafted material, the periodfrom March to June is favorable (Vers-choore-Martouzet, 1985).

Regarding the meristems, Verschoore-Martouzet (1985) found no contaminationafter using calcium hypochlorite, irrespec-tive of the date of removal and origin of theexplant in an 80-yr-old tree. Walker (1986)also found none, even without using disin-fection treatment.

Multiplication phase

Explants used were stem segments (fig1 a), bearing 5 to 8 leaves. The MM culturemedium used during the multiplicationphase was derived from that of Murashigeand Skoog (1962) with auxin and cytokininadded. The media used by different au-thors have been summarized by Boulay(1989). In our laboratory, the MM multipli-cation medium used contains BAP (2.2 10-6M) and NAA (5 10-8 M) (tables I, II). Theperiod of culture on MM varies from 3 to 8weeks (Boulay, 1978; de la Goublaye,1981; Fouret, 1987; Tranvan et al, 1991).

After axillary buds had developed (fig1b), newly formed shoots were isolatedand transferred to identical fresh mediumfor intensive multiplication. They can betransferred to an EM elongation medium(fig 1 c), and the obtained stems (fig 1 d) di-vided for multiplication on MM.

The material can be grown under weak

lighting and a wide range of temperatures,with a preference for temperatures of&ap; 24°C.

A multiplication ratio from 3 up to 8 canbe achieved at 3 weekly intervals (Boulay,1985). The ability of the explants to devel-op axillary buds depends on the chrono-logical age of the mother plant and theoriginal shoot, and on the original positionof the material on both the ortet and ramet

(Boulay, 1978; de la Goublaye, 1981;Verschoore-Martouzet, 1985). Thus, ex-

plants originating from a sucker are morereactive than explants derived from the

crown, and for the same sucker the most

apical regions (most recently formed)show the best response.

Elongation phase

An isolated cultured meristem will elon-

gate into a leafy shoot if transferred to amodified Murashige and Skoog (1962) me-dium without added growth regulator, and

without activated charcoal (Walker et al,1985).

But more generally, axillary buds whichdevelop during the multiplication phase (fig1b) are isolated and transferred to the EMmedium (fig 1 c) based on the Murashigeand Skoog (1962) formula, again withouthormones, but containing activated charcoal(tables I, II). The favourable effect of activat-ed charcoal on growth of Sequoia sempervi-rens in vitro has been previously reportedby Boulay (1978). The time of culture on EMcan vary from 4 to 8 weeks (Boulay, 1978;Fouret, 1987; Tranvan et al, 1991).

The behaviour and the morphology ofthe shoots differ over time according to thephysiological state of the material intro-duced into culture. This state depends pri-marily on the original position on the moth-er plant and its chronological age: thus dela Goublaye (1981), comparing the behavi-our of 3 topoclones (top and base of thecrown, base of the trunk) of clone S5 froma tree aged at least 500 yr (Franclet,1981), observed that after 1 subculture thetopoclone from the top of the crown grew

lowest. Later Fouret (1987) noticed thatthe growth of materials cultured in vitro forseveral years was distinctly slower in a

clone (No AFOCEL 78461 = clone II in our

laboratory) from a 500-yr-old tree (ARC154) than in clones from a 1-yr-old (NoAFOCEL 83753 = clone J) or 50-yr-old (NoAFOCEL 77304 = clone I) trees.

Fouret (1987) reported that, at the endof the elongation phase, when the initialmaterial was young, the leaves were long,soft and light in colour. Furthermore, thephyllotaxy was of axial type in a clone froma 1-yr-old tree, whereas it was either of ax-ial or crosswise-opposite type in a clonefrom a 50-yr-old tree, and of crosswise-

opposite type or more often distichous typein a clone from a 500-yr-old tree.

After 2 months on EM medium, stemssometimes rooted spontaneously althoughwithout any auxin, but no rooting was ob-served when the material originated fromthe very old tree (Fouret, 1987).

After a culture on EM of > 3 months the

stems from clone II sometimes showed ba-sal or apically situated outgrowths fromwhich slender shoots might grow (Bek-kaoui et al, 1984; Fouret, 1987).

If the period of culture on EM lasted for4 months or longer (Tranvan, unpublishedresults) the shoots of the old material

(clone II) (fig 2a) rooted spontaneously un-less they stopped growing and their apicalbud was getting round (fig 2b), after which,if growth took up again (fig 2c,d), very mor-phologically different areas were observedto have developed along the stem.

Effect of repeated subcultures

Although during repeated subcultures ac-cording to the MM-EM sequence the ma-terial became increasingly reactive (Bou-lay, 1985), differences in reactivitycorrelated with the position of explant ori-

gin and persisted for a long time. Thus ma-terial of sucker origin yielded better resultsthan the inferior branches of the crown andbetter still than the superior branches (de laGoublaye, 1981). Likewise, the morphologyof shoots from the old material (clone II) re-mained different to that of shoots from the

young material (clone J) for several months(Fouret, 1987). But after 4 yr of repeatedsubcultures the clone originating from a

shoot of the top of the 80-yr-old NP29 tree(Verschoore-Martouzet, 1985) behaved in

morphology and reactivity like the clone ob-tained from the sucker of the same tree

(Franclet, personal communication).Verschoore-Martouzet (1985) observed

that, with NP 29 tree material, shoots de-veloped from stem segments after 3 or 4subcultures on medium supplemented withcytokinin had orthotropic growth, whereasthe shoots which developed initially in vitrowere plagiogropic.

Micropropagation using passagesthrough EM (-MM-EM- sequence) avoidsthe abnormalities of waterlogging and fas-ciation, which often appear in protocols in-volving several subcultures on cytokinincontaining medium (Fouret, 1987; Boulay,1989) and increases shoot production.

Rooting and acclimatization phases

The shoots obtained in vitro can either be

rooted directly ex vitro under horticultural

conditions, or rooted in vitro prior to accli-matization ex vitro.

Rooting ex vitro

In the first studies, shoots could not berooted in vitro, but had to be rooted ex vitro(Boulay, 1978). Later, Poissonnier et al

(1981) proposed the use of cold storage ofthe in vitro shoots and use of specific sub-strates to improve rooting.

The improvement of shoot productionand the improvement of the ex vitro rootingafter repeated subcultures enabled > 200clones originated from adult trees selectedin various regions of France and Californiato be multiplied (Franclet et al, 1987).These cuttings were used for the establish-ment of a mother tree orchard at Guingampin Brittany, from which orthotropic shootscould be recovered in the longer term bypruning and cutting back operations forcommercial production of cuttings.

After observing effects on conventionalpropagation of the original location of thecuttings on adult trees, de la Goublaye(1981), using an 80-yr-old tree located atFontainebleau, studied the effects of to-

pophysis on shoot growth habit in vitro

during repeated subcultures according tothe MM-EM sequence. She found therewas a progressive improvement in rootingspeed (ex vitro) and, more slowly, in the

recovery of orthotropy. However, the topo-physical effects did not totally disappearover the period of the subcultures. Withone of the topoclones of the Fontainebleautree the number of subcultures not only af-fected recovery of orthotropy but also laterwood productivity ex vitro (Franclet, un-

published results). A similar result was ob-tained in an experiment conducted on ma-terials from a 500-yr-old tree, originatingfrom California.

But despite this progress, the problemsof long rooting initiation period, rooting fluc-tuations according to season and plant ma-terial used, persistant plagiotropic growthremained. Consequently studies were un-dertaken on the effects of in vitro rooting.

Rooting in vitro and acclimatization

In initial experiments with materials fromsprouts of different aged trees, Boulay(1978) obtained an in vitro rooting rate of 5to 25% depending on the media used,

which were various dilutions of modified

Murashige and Skoog (1962) medium withan addition of auxin. Using materials fromsprouts of adult trees, Ball et al (1978) ob-tained = 20% of rooted shoots with an or-

thotropic growth habit after acclimatization,although it has to be emphasized that thesprouts used were physiologically youngmaterial (see Rejuvenation).

In an attempt to optimize in vitro rooting,Bekkaoui et al (1984) studied the behaviorof material from 2 clones (I and II) obtainedfrom a 50 and 500 year-old tree respec-tively, using multiplication and elongationtechniques similar to those of Boulay(1978). The apical microcuttings removedafter the elongation phase were 1 to 2 cm

long and had 8 leaves. Optimal conditionsfor rooting were found to be: rooting induc-tion (1 wk) on a RIM medium supplement-ed with auxin (NAA 5 10-5 M); rooting ex-pression (6 wk) on root expression (REM)medium, identical but without auxin (tablesI, II) (temperature: +20 to 25°C); daily illu-mination: 9 h (100 W m-2) for clone I; 9 h(100 W m-2) + 15 h red (12 to 15 W m-2)for clone II). There was a very distinct dif-ference in reactivity between the 2 clones,with the percentage of rooted explants andthe mean number of roots per explant al-ways inferior in the oldest material. Thebest results obtained were in clone I, 90%rooting, with a mean of 6 roots per explant;in clone II, 60% rooting and mean of 3 rootsper explant. The material from the 500-yr-old tree was also more limited in its ability toadapt to the variety of in vitro culture condi-tions used. Rooting ability and maintenanceof this property under widely varying envi-ronmental conditions appeared to charac-terize juvenility.

In a further work on rooting Walker et al(1985, 1987) applied the following treat-

ments to microcuttings from clones ob-tained from young and old trees: a root in-duction phase under dark conditions (5 d);an expression phase (35 d) under different

types of lighting; an acclimatization phaseunder natural light, with additional lightingto give a 16 h photoperiod. For the youngeasily rooting material, acclimatization wasaccelerated when rooting was obtainedunder a high quantum flux (up to 280 &mu;Em-2·s-1). The old material was difficult-to-root, but a high quantum flux increased therooting rate and number of roots. Undershort days, night breaks of red or far redlight had no effect. For the older clone ac-climatization also depended on the periodthe rooted shoots were cultured on agarmedium. After 10 d they could be acclima-tized with 70% success rate.

The practice in our laboratory (Fouret,1987; Fouret et al, 1989; Tranvan et al,1991) has been to take apical cuttings 6cm in length from material maintained in vi-tro for rooting induction in short days afterculture on EM (induction: 1 wk; expression:6 wk) (fig 3). If the culture period on EMexceeds 3 months, the apical cuttings pro-gressively lose their ability to root

(’experimental aging’; Tranvan: unpub-lished observations). For clone II the num-

ber of subcultures has affected in vitro

rooting (Arnaud et al, 1987). At present, af-ter 12 yr of repeated in vitro culture, shootsfrom clone II often root spontaneously.

REJUVENATION

Different kinds of aging occur in trees (For-tanier and Jonkers, 1976; Chaperon,1979). Chronological age is the duration oftime since germination. Physiological ag-ing reflected, for example, in rooting ability,is the result of the increase in tree size and

complexity. Ontogenetic age reflects thesuccessive phases in development, re-

vealed for example in topophysis (Seeling-er, 1924; Franclet, 1983; Boulay, 1987b).

Rejuvenation is a necessary prerequisitefor mass propagation, and can be defined

as the recovery by old plant material of atleast some of the properties of younger ma-terial (see Pierik, 1990). Walker (1985) pre-fered the expression ’rejuvenilization’, re-

serving the expression ’rejuvenation’ for therapid and total recovery of juvenile charac-ter, for example in apomictic or zygotic em-bryo formation (Franclet and Boulay, 1989).

In situ pruning, cutting back or graftingcan induce a rejuvenating process (Ver-shoore-Martouzet, 1985), improving in vitroperformance (Franclet, 1981; Franclet et

al, 1987). The effect of these techniques isto bring the root system closer to theabove ground shoot system (Doorenbos,1965; Chaperon, 1979; Franclet et al,1980; Favre, 1980). The role of roots couldbe to supply cytokinins (Kende, 1964; Itaiand Vaadia, 1965; Sitton et al, 1967).

Media containing a cytokinin appear tohave a rejuvenation effect (Boulay, 1978;de la Goublaye, 1981). For example, sub-cultures on a medium supplemented withcytokinin increases the K/Ca ratio in mat-

erials from an 80-yr-old tree to values char-acteristic of seedlings. Similarly, the peak’peroxidase activity/total proteins’ increas-es with the number of subcultures on cy-tokinin-containing medium (Verschoore-Martouzet, 1985; Boulay, 1987b). Theseratios are correlated with rooting ability.These results showed that it was logical toconsider the in vitro culture as a techniqueutilizable for attempting to rejuvenate plantmaterial. In addition, this was suggestedby Franclet (1981), Nozeran et al (1982),Margara (1982) for various materials.

Several protocols were developed witha double aim:

- to study the conditions and mechanismsinvolved in the rejuvenation process (basicaim);- to attempt the recovery of all ’juvenilityproperties’, particularly the orthotropy in

material issued from adult or very old trees

(applied aim).

Single or repeated cultures of isolatedcaulinary meristems (protocol IM)

Meristems (0.1 mm shoot tips, includingthe meristematic dome with 1 or 2 leaf pri-mordia) were removed (Verschoore-Martouzet, 1985) from an 80-yr-old tree atdifferent periods of the year from shootssituated at the base of the tree or at 15 m

height from suckers, or from the same or-gans grafted onto seedlings, and from

seedlings from the same tree. The shootsdeveloped from these meristems were

found to recover some juvenile charactersafter culture on a modified Murashige andSkoog medium with IAA (Boulay, 1989): egtypical juvenile morphology, good rootingability, partial recovery of the orthotropy.The time of year of removal had little influ-

ence, but ’memory’ of topophysis was noteradicated.

With repeated subcultures of meristemsissued from a very old tree (ARC 154: 500yr), the shoots progressively recovered ayoung physiological state, evaluated by abetter rooting ability (realized ex vitro), bythe acquisition of the ability for adventitiousbudding on isolated leaves and by a betterreactivity* of the isolated apical meristems(Walker, 1986). But the plants still main-tained plagiotropic growth in the green-house. Total (according to the praticians)or ontogenetic (see Schaffalitzky de Muck-adell, 1959) rejuvenation of material from a

very old tree has thus not yet beenachieved.

Micrografting of apicesfrom old materialonto young rootstocks (protocol MG)

The effects have been studied of graftingaged axillary branches apices from unroot-ed shoots of clone II onto vitroplants of theclone J (Tranvan et al, 1991). The shootsproviding the apices were maintained onthe EM medium (elongation medium) ofthe MM-EM sequence for 2, 4 or 6months. The apex (= 4 mm) was graftedonto an oblique section of the rootstock at1 cm distance from the roots (fig 4a).When the grafted apices grew, the

scions acquired a growth rate and mor-

phology similar to those of clone J (fig 4b).Shoots were then excised and submittedto a root inducing treatment according toBekkaoui et al (1984). Micrografting ap-peared to increase the number of shootswhich rooted, speed of rooting and qualityof the root system (fig 4c). After transplant-ing ex vitro the rooted stems had an ortho-tropic growth habit for several weeks, butthen reverted to plagiotropy. However, avery small number of plants were still or-

thotropic 2 yr later (fig 5).Thus micrografting initiated a rejuvena-

tion process. This technique is currently

* The ’meristem test’ is based on the number of days necessary ’to get the cultured meristem greenagain’.

being improved in our laboratory (Tranvan,unpublished results). The efficiency of thistechnique was confirmed by Huang et al(1992) who studied morphological and pro-tein markers of rejuvenation status.

Repeated subcultureof stem segments (protocol RS)

Fouret (1987) and Fouret et al (1985,1986a, 1989) used stem segments with 3leaves (thus 3 axillary meristematic areas)from different-aged trees (1 yr (clone J),50 yr (clone I) and 500 yr (clone II)) to

study the effect of repeated subcultures.The explants were cultured on MM medi-um containing BAP (2.2 10-6 M) and NAA(5 10-8 M) or on MO (same medium asMM but without growth regulator). Eight

protocols were tested, differing in frequen-cy of subculturing on 2 different media andin sequence of using these media. Efficien-cy of rejuvenation of each protocol wasevaluated for 18 wk after the start of the

culture considering the modifications in a

number of properties (Fouret et al, 1985;Franclet et al, 1987): improvement of

growth activity of the longest axillary stem,reappearance of a young type phyllotaxy,reduction of the reactivation time of the iso-lated caulinary meristems, increase in therate of rooted explants and the reduction ofrooting time (Fouret et al, 1985), increasein mean root number per explant, improve-ment of the quality of the root system. In

addition, we observed that the increase ofthe endogenous IAA/ABA ratio was corre-lated with rooting ability of the shoots (Fou-ret et al, 1986b; Fouret, 1987) and another

criterion was the ability to produce adventi-tious buds on isolated leaves (Fouret,1987). The total or ontogenetic rejuvena-tion is considered as successful when the

vitroplants exhibit continuous orthotropicgrowth after ex vitro transfer (Fouret et al,1989).

Rejuvenation was more difficult to ob-tain in clone II than in clone I. The resultsshowed the prime importance of the ’fre-

quences of the subcultures’ for the rejuve-nation of old material. Frequence of ’onesubculture per week’ was most efficient,but it was also beneficial to apply a rejuve-nation-inducing treatment (3 transfers onMM for example), and to conduct the othertransfers on MO.

In this work, the juvenile properties ap-pear successively more or less rapidly andmore or less clearly according to the proto-col and the physiological state of the initialmaterial. But certain properties of the reju-venated state may disappear (Arnaud et

al, 1989). The rejuvenation - ’rejuveniliza-tion’ according to Walker (1986) - appearsas a sequence of ability states. The

cuttings from the material rejuvenated ac-cording to the RS protocol kept a plagio-tropic growth habit ex vitro.

Adventitious budding induced on isolat-ed leaves of previously rejuvenated materi-al in one of the protocols produced plants(Fouret et al, 1989) which maintained exvitro orthotropic growth for 4 yr in the

greenhouse and then outdoors. It has

been recently observed (Monteuuis-Bon,unpublished results) that the clonal materi-al produced by these epiphyllous buds

contained a specific protein profile. Onto-genetic rejuvenation has thus been ob-tained in clone II from a 500-yr-old tree(ARC 154) with this specific protocol. How-ever, in such experiments it is difficult to

distinguish the contribution to shoot growthmodalities of material rejuvenation, root

system morphology and environmental

conditions during the acclimatization pro-cess which can greatly influence root sys-tem quality. How such rejuvenated materi-al will grow in the long term is uncertain.One of the first trees produced by epiphyl-lous budding (during the IM protocol of

Walker, 1986) is 5.2 m tall after 5 seasonscompared to 0.70 m for the control ob-

tained using the technique of Boulay(1978) (Franclet, unpublished results).

These results indicate that several fac-

tors control the rejuvenation process: therelease of shoot meristems from the correl-ative control, the repeated subcultures onmedium supplemented with cytokinin, andthe closeness of the shoot meristem to the

root system.

IN VITRO REGENERATION

Horry-Charrier (1988) isolated protoplastsfrom foliar tissues of vitroplants in youngmaterial and produced microcalli of = 30

cells, but without regeneration. Regenera-tion has, however, been obtained by othermethods.

Formation of somatic embryos

Bourgkard and Favre (1988, 1989) ob-

tained somatic embryos in calli on mature

zygotic embryos and on cotyledons andhypocotyl segments isolated from

vitroseedlings 7 d after germination. Thisembryogenesis was only observed whenseeds recently collected or stored in a coldroom for < 11 to 12 months (darkness;+4°C). Embryogenic potential in regularlysubcultured calli was maintained for only12 wk. In this embryogenesis, no proem-bryo was observed, and the somatic em-bryos with typical bipolarous structure

were obtained in darkness on culture medi-um containing KIN 2 &mu;M, BAP 2 &mu;M and

2.4-D 2.5 &mu;M, without a sequence of me-dia of different composition. The callus ob-tained was solid and consistent in charac-

ter, and the somatic embryos were mixedwith adventitious buds. On a medium with-out hormone and under light, the somaticembryo shoots grew, but most of their rootsturned brown and developed lateral roots.

In our laboratory (Lefrançois, 1988),neoformed buds (fig 6) (cf Formation ofadventitious buds) were isolated from callifrom mature zygotic embryos and placedon elongation medium. These buds devel-oped into leafy stems (fig 7), and, from the&ap; 12-cm stems, the ten 1- to 3-cm longsubapical leaves were removed and putthrough a double treatment consisting ofcaulogenetic then callogenetic media

(Krogstrup, 1986; Lelu, 1987). On one

leaf, a soft translucent white callus wasformed which contained very numerous

polarized structures (fig 8). Each structurewith 2 parts, one with a meristematic cellcluster and the other with long vacuolizedcells, is similar in appearance to young zy-gotic embryos (Singh, 1978). These struc-tures were also similar to the somatic pro-embryos observed in other gymnosperms(Gupta and Durzan, 1986; Hakman andFowke, 1987; Lelu et al, 1987).

Formation of adventitious buds

Female gametophytes

Plantlets have been regenerated from cul-tured female gametophytes of Sequoiasempervirens (Ball, 1981, 1987). Matureseeds were excised from green cones

which were not yet opened. The cones andthen each individual seed were surface-sterilized by soaking in full-strength bleach(Ball, 1987). After removal of testae, thethin layer of female gametophytes (naturallyaxenic) was isolated from the embryo. After= 2 months on the culture medium, a callus

extruded from the bottom of the gameto-phyte cavity, where the embryo used to be,with some buds already present. After 6months’ culture (with transfers at monthly in-tervals) the callus was covered with shoots,which could be removed and grown on oth-

er media into orthotropic rooted plants.Many of them have subsequently devel-

oped into small trees (Ball, 1987). The chro-mosome number of the female gameto-phyte derived plants was 66. Chromosomecounts of living female gametophytes ofseeds have given various counts; it is as-sumed that the occasional diploid cells di-vide to produce the callus from which thebuds regenerated (Ball, 1987).

Mature zygotic embryos andisolated cotyledons of these embryos

Using a protocol similar to that of Lelu et al(1987) with mature embryos and isolatedcotyledons, organogenous calli have beenfound to develop with caulinary neoforma-tions, and in a lower amount with root neo-formations (Chiffaudel and Stroobants,1987). Vascular connections pointed be-tween the caulinary neoformations and thecallus tissues. According to Haccius

(1978), these buds were adventitious budsand not somatic embryos whose radicularpole would have aborted. After transfer toelongation medium, these buds developedleafy stems, rooting either spontaneouslyor after induction (RIM-REM treatment).More recently, Tranvan (unpublished re-

sults) has observed that adventitious budscan develop on isolated cotyledons withouta callus stage (fig 9).

Bourgkard and Favre (1988, 1989) alsoobtained compact organogenic callus onmature entire zygotic embryos or dividedyoung seedlings in which adventitiousbuds were mixed with somatic embryos,and observed that caulogenesis de-

creased, as did embryogenesis, with theduration of seed storage at + 4°C.

Stem segments

Ball (1950) obtained callus capable of re-generating buds from stem segments is-sued from burls (this was the first in vitro

culture in Sequoia sempervirens) and in

1978 Ball et al obtained numerous adventi-

tious buds from juvenile and orthotropicstem segments from the base of an adulttree. These buds developed from corticalproliferating tissues through ruptures in theepidermis into orthotropic shoots which

could be rooted and transplanted in the

greenhouse. By contrast, plagiotropic stemsegments from the crown formed few ad-ventitious buds, and these buds only grewslowly and never produced acclimatized

plants.Ball et al (1978) and Ball (1987) ob-

served that Wolter and Skoog’s mineralmixture was better than Murashige andSkoog’s medium for regeneration fromstem explants (growth regulators were

2.4.5-T and SD 8339). After elongation ona medium lacking cytokinin and containingactivated charcoal and auxin, the shoots

were transferred into rooting medium I(with IBA) for 12 h, and then to rooting me-dium II (without auxin and with activatedcharcoal). After 30 d, shoots were trans-ferred to a mixture (2:1:1) of sphagnummoss perlite vermiculite in tubes irrigatedwith a mineral solution. The tubes were

stored in a greenhouse. Ball (1987) ad-vised that a large root system should beestablished before plantlets were trans-

ferred to soil.

Leaves

Ball et al (1978) and Ball (1987) observedthat isolated leaves, cut transversely in

half, and laid on culture medium supple-mented with cytokinin SD 8339 and auxin2.4-D, required an intervening callus stageproduced on the section surface for regen-eration of buds. Nevertheless, adventitiousbuds have been produced without appar-ent proliferating callus by:- maintaining in vitro basal leaves from

young material (clone J) or, in clones J, Iand even II, on basal leaves of the stem incontact with the culture medium during RSrejuvenation protocols (Fouret, 1987);- after specific treatment of subapical(young) isolated leaves from clone II

during the rejuvenation protocols IM, Walk-er, 1986), RS (Fouret, 1987; Fouret et al,1989) or MG (Tranvan, unpublishedresults) (fig 10). The epiphyllous bud

inducing treatments consisted of 2 stages:induction (3 wk) on BM medium (similar toMM but without NAA and with BAP 1 mg·

l-1) and then expression (4 wk) on the

BMO medium (similar to BM but withoutBAP).

Sometimes elongation of such buds is

difficult to achieve (Tranvan, unpublishedresults). Nevertheless the leafy shoots,which grew from epiphyllous buds inducedon material of clone II and rejuvenated ac-

cording to the type SR protocol (Fouret etal, 1989), rooted easily in vitro after induc-tion and acclimatized easily. They had themorphology of young plants, were vigorousand showed orthotropic growth (fig 11) (cfRepeated subculture of stem segments(protocol RS). By contrast, the non-

rejuvenated material of the same clone re-mained plagiotropic (fig 12), and excep-tionally produced male cones on some

plants only 21 months after transplanting inthe greenhouse (fig 13) (Tranvan, unpub-lished results).

The adventitious budding technique hasgreat potential for multiplication, but the

buds obtained after a callusing stage maynot be true-to-type genetic, as Patel andBerlyn (1982) have observed for Pinuscoulteri. However, Ball (1987) countedchromosomes in root-tips of Sequoia sem-pervirens trees regenerated from stem

pieces and leaf inocula and found that inboth cases the plants are regenerated onlyfrom diploid cells.

RECOMMENDED PROTOCOLFOR THE MICROPROPAGATIONOF SEQUOIA SEMPERVIRENS

Regeneration protocols have been detailedby Ball (1987). Described below are ourrecommended variants to the protocol pro-posed by Boulay (1989).

Culture establishment

Choice of initial explant

Stem pieces should be taken from basalsprouts of an old tree or from activelygrowing grafted plants grown in the green-house. If the explants are taken from thetop of an adult or old tree, July is the bestperiod to initiate sterile culture.

Disinfection and sterilization

The stem segments should be soaked in

benlate solution (&ap; 0.2%, about 24 h) or inliquid soap (2 or 3 min), rinsed in waterand then ethanol at 70° GL (1 or 2 min),soaked in a commercial sodium hypochlor-ite solution (5 to 20 min), and finally rinsed3 times with sterile distilled water.

The duration of these treatments and the

concentration of the solutions used depend

upon the season, environmental conditionsof the mother plant, age, situation on thetree and lignification status of the explant.

Choice of miniaturized explants

It is recommended to use small, little or

non lignified stem segments (&ap; 15 mm inheight with some leaves); or preferablycaulinary apices (meristem surrounded bysome foliar primordia).

Multiplication

Stem segments or meristems are placedin or on multiplication medium (MM) whichis composed of half-strength Murashigeand Skoog medium as modified by Boulay(1978), NAA (0.01 mg·l-1), BAP (0.5 mg·l-1), sucrose (30 g·l-1) and agar (7 g·l-1).

Elongation

After 1 or 2 months, the axillary buds (orthe very short axillary stems) should betransferred to an elongation medium (EM),similar to MM but without growth regulatorand containing activated charcoal (20 g l-1).

Micropropagation is achieved by usingsections (5 or 6 leaves) of vitroshoots, el-ongated on EM for 1 or 2 months, for cul-ture on MM (several MM-EM cycles).

Rooting and acclimatization (weaning)

Ex vitro rooting and weaning

It is recommended to harden the vitro-shoots for 1 or 2 wk at a temperature of 5to 10°C under natural light (10-13 h illumi-nation). The cuttings should then be

soaked for = 24 h in an auxin solution andtransferred to a heated rooting substrateand maintained under a mist system or en-closed in a propagator (photoperiod: 16 h).

After 6 to 8 wk, the cuttings should betransferred to Melfert ’envelope’ and theaerial parts acclimatized by progressive re-duction of relative humidity.

In vitro rooting, transfer ex vitroand acclimatization

Terminal vitrocuttings &ap; 6 cm high shouldbe taken from shoots elongated on EM.

Rooting induction is achieved by culture onone-third strength Murashige and Skoogmedium as modified by Boulay (1978),with NAA (9.3 mg·l-1) (RIM) for 1 wk. Root-

ing expression is achieved on a similar me-dium without auxin (REM).

Two methods can then be applied:Method A: Approximately 30 d after the ap-pearance of the first roots, the vitroplantsare transferred ex vitro to a perlite/vermiculite mixture watered with a mineral

nutrient solution (for example, Phytotronsolution) (de Bilderling and Lourtioux,1976) or AFOCEL solution (Poissonnier etal, 1981) and then transferred to a horticul-tural substrate.

Method B: Approximately 10 d after the ap-pearance of the first roots, the vitroplantsare maintained at cool temperature (eg22°/12°C) under natural light for 1 wk; theyare then transferred ex vitro, enclosed in aMelfert ’envelope’ and placed on a thickmoist cloth maintained at 22°C by bottomheat. When roots are visible in the contain-

er, the plants are transferred to a horticul-tural substrate.

In both cases, the plastic tent must begradually removed in order to progressive-ly reduce air moisture.

Rejuvenation

If rooting of the material is difficult, motherplant material should be submitted to a re-juvenation protocols prior to root inductiontreatment. Three kinds of in vitro rejuvena-tion protocols can be used: 1) single or re-peated culture(s) of isolated caulinary mer-istems; 2) single or repeated micrografting(s) of apices from old material onto youngrootstocks; 3) repeated subcultures of

stem segments.If the vitroplants remain plagiotropic af-

ter transfer ex vitro it is recommended tosubmit the mother plant material to a reju-

venation protocol leading to epiphyllousbudding. The shoots produced from the

epiphyllous buds can then be induced toroot, or introduced onto the MM-EM cycle,before rooting induction.

PRACTICAL APPLICATIONS

These techniques have been in continuouspractice for &ap; 14 yr, some with clones fromvery old trees (for example ARC 154, ARC28).

Boulay (1987a) has reported the AFO-CEL micropropagation techniques for theproduction of rooted clones from 120 se-lected adult genotypes with a view to refo-restation.

The technique of Boulay (1978), later

modified in our laboratory, involving alter-nate subcultures of = 4 to 8 weeks on amedium containing an auxin and a cytoki-nin, followed by a medium lacking growthregulator but containing activated charcoal,allows the vitrostems of Sequoia to bemaintained and to multiply for period of

years without the ’clonal degeneration’found in Douglas fir (Bekkaoui et al, 1986).

By using this technique the rapid multi-plication of > 400 clones of old trees se-lected by AFOCEL scientists in parks andgardens of Western Europe (France, Eng-land, Ireland, Germany and Belgium) hasbeen effected. It has also facilitated the in-troduction to Europe of almost all 200clones of the ’Kuser collection’ (a completesampling of the ’genetic pool’ of Sequoiasempervirens in its natural area). It has en-sured the multiplication of the conservationcollections and accelerated the establish-ment of planting for clonal evaluation at nu-merous sites in France.

Some historical or ornamental trees

have been commercially propagated usingmicropropagation eg by RMCV (Re-cherche, Multiplication, Contrôle des Végé-

taux Co) in France. In the USA the Compa-nies Simpson Timber and Georgia Pacificproduce vitroplants (Boulay, 1987a) follow-ing a scheme initiated by Ball et al (1978)or adopting the technique of Boulay(1978).

PROSPECTS

The interest of Sequoia sempervirens is

recognized in the forestry and paper indus-try. But future prospects for large-scale mi-cropropagation for reforestation purposeswill depend upon economic considerations(the present methods are still too expen-sive for mass production) and increasedbasic knowledge of rejuvenation process-es. Concerning this last point, the Sequoiasempervirens is considered as good exper-imental material for studying rejuvenationin woody species (Fouret et al, 1986a;Boulay, 1987b; Franclet et al, 1987). Thesearch for specific gene expression asso-ciated with rejuvenation has already started(Bon-Monteuuis, unpublished results;Huang et al, 1992). Moreover, the control ofsomatic embryogenesis, associated or notwith transformation experiments, constitutesan extremely interesting task for the future.Sequoia sempervirens can be consideredas a good model for the application of thesenew biotechnologies to woody species withthe aim of improving the production of ge-netically selected or modified trees.

ACKNOWLEDGMENT

We would like to thank Pr E Migniniac for hissuggestions during useful talks.

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