Plant Cell, Iissue and Organ Culture 37: 287-295, 1994. (~) 1994 Kluwer Academic Publishers. Printed in the Netherlands.

Forced flushing of branch segments as a method for obtaining reactive explants of mature Quercus robur trees for micropropagation

A n a M. Viei tez, M. C o n c e p c i 6 n Sfinchez, Juan B. A m o - M a r c o 1 & A n t o n i o Bal les te r Plant Physiology, CSIC, Apartado 122, 15080 Santiago de Compostela, Spain; 1Department of Biology, Faculty of Biology, University of Valencia, Spain

Received 16 September 1993; accepted in revised form 19 March 1994

Key words: horizontal culture, in vitro tissue culture, oak, reculture, reinvigoration

Abstract

The aim of this study was to micropropagate mature Quercus robur L. trees when material retaining physiologically juvenile characteristics (stump sprouts, epicormic shoots) is not available. Branch segments from 70-300 year-old trees were force-flushed and the flushed, partially rejuvenated or reinvigorated shoots were used as a source of explants for establishment of cultures. In vitro establishment and multiplication was achieved with seven of the eight selected trees. The proliferation capacity of cultures of vertically placed explants declined after several subcultures, but efficient shoot multiplication was achieved by culturing decapitated shoots placed horizontally on GD medium supplemented with 0.89 ~tM of 6-benzyladenine. Reculturing the same horizontal explant several times allowed both higher multiplication rates and a shorter subculture cycle (2 weeks). An initial dark period of 5 days generally improved rooting capacity, which ranged, depending on clone, from 15 to 46%.

Abbreviations: BA - 6-benzyladenine, GD - Gresshoff and Doy Medium, IBA - indole-3-butyric acid

Introduction

Micropropagation contributes to forest tree improve- ment by multiplication of genotypes of proven value. Ease of vegetative propagation tends to diminish as trees approach a size allowing reliable evaluation of their desirable qualities; explants from adult trees are notoriously unreactive (Bonga 1987). In some cas- es, micropropagation is facilitated because the tree produces tissues with juvenile characteristics, such as stump sprouts, epicormic shoots or root suckers.

Oak plantlets have been generated in vitro from basal shoots or stump sprouts of mature trees (Vieitez et al. 1985; San-Jos6 et al. 1988; Chalupa 1988), but it is difficult to establish in vitro cultures from buds of the current season's growth in the crown (S~mchez 1991; Vermeer et al. 1991). When suitable material taken from juvenile parts of the tree is not naturally available, it is necessary to apply pretreatments that will rejuvenate or reactivate mature material for sub- sequent micropropagation.

Methods often recommended for reinvigoration (reversal of aging, Pierik 1990) or partial rejuvena- tion (reversal of maturation), include repeated grafting, serial cutting propagation, severe pruning, hedging and the use of stool beds (in spite of being time consuming, frequently impracticable, or failing to leave most of the tree intact). Hedging and stool bed methods make use of preformed dormant buds that remain quiescent after early initiation; the outgrowth of these buds often leads to the development of juvenile shoots, partic- ularly when they arise in the so-called juvenile zone at the base of the tree (Franclet 1977). Similar shoots arising higher up the tree (epicormic shoots) most- ly originate from suppressed accessory buds, but can also be of adventitious origin (Evers et al. 1990). The manipulation of stock plants through sectioning the trunk to induce epicormic shoots with greater juvenil- ity is another rejuvenation method (Evers et al. 1990). A variant of this technique, which does not destroy the tree, consists of sectioning of thick branches of the tree to induce the flushing of epicormic shoots in

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the branch sections in a greenhouse or growth cham- ber. This method has been used for in vitro culture of explants of mature trees of Eucalyptus grandis (Ike- mori 1987).

In this study

- we explored the feasibility of in vitro establishment and multiplication of cultures derived from old Quercus robur trees, to which end we force-flushed branch segments and used the flushed shoots as a source of explants for the establishment of the cul- tures, and

- we improved the efficiency of shoot multiplication by obtaining a stabilized shoot multiplication stage by in vitro reinvigoration through repeated subcul- turing.

M a t e r i a l s a n d m e t h o d s

Initiation of cultures

Old branches, 3-5 cm thick, were collected from the crown of 700 to 300-year-old Quercus robur trees des- ignated SL1, SL3, Salnza, Monasterio, Tixon, Pega, San Esteban and F1. These branches were collected at the end of March, just before the trees were about to burst into growth, and cut in 25-30 cm segments. The branch segments were laid horizontally on moist perlite beds and forced to flush accessory or epicormic shoots in a growth cabinet with a temperature of 24°C, a 16-h photoperiod and a relative humidity of 80-85%. After 2-3 weeks the flushed shoots (2-10 cm long) were used as the source of explants. Flushed shoots were stripped of leaves and surface-disinfested by suc- cessive immersion for 30 sec in 70% alcohol and 9 min in 0. 8% sodium hypochlorite with 1 ml 1-1 of Tween 80. This was followed by three rinses in ster- ile distilled water. The explants, consisting of 5 mm shoot tips and nodal segments, were placed upright in 20 × 150 mm test tubes containing 15 ml of ini- tial medium. This consisted of GD (Gresshoff & Doy 1972) medium supplemented with 4.4 laM BA, 30 g 1-1 sucrose and 6 g 1-1 VitroAgar (Hispanlab S.A.). The pH of the medium was brought to 5.5-5.6 prior to autoclaving at 121°C and 108 kPa for 20 min. One day after implantation in vitro, the explants were moved to the opposite side of the same test tube to separate them from excreted phenolics. After 6 weeks in initial medium, with transfer to fresh medium every 2 weeks, the following data were recorded:

Fig. 1. Forced flushing of a crown branch segment from a 100-year-old Quercus robur tree (Clone SL1). Bar = 2.7 cm

- i n vitro reactivity, defined as the percentage of explants with shoot development;

- the number of shoots longer than 6 nun per reactive explant; and

- t h e length of the tallest shoot on each reactive explant.

In a first experiment two approximately 100-year-old trees (SLI and SL3) were used. For both trees, the in vitro reactivity of explants taken from shoots flushed on old branch segments was compared with that of explants from shoots flushed in the growth cabinet from twigs of the previous year's growth collected in March and of shoots of the current year's growth collected in April. Subsequently, cultures were also initiated from old branch segments of the six other adult trees aged 70-300 years (see Table 2).

All cultures were kept in a growth chamber with a 16-h photoperiod (30 ~tmol m -2 s -1, Cool-White fluorescent lights) and 25°C day/20°C night tempera- tures. These incubation conditions were also applied to the shoot multiplication and rooting stages described below.

Shoot multiplication and rooting

The new shoots obtained in vitro were excised and subcultured to produce clonal shoot multiplication cul- tures. At first, the multiplication explants were placed upright in the multiplication medium (initial medium with the concentration of BA reduced to 0.89 ~tM). Since shoot growth and multiplication rates decreased after several subcultures, horizontal explant position was tried as a means to invigorate shoot growth (Vieit- ez et al. 1993). Shoot explants with the apical 2 mm removed were placed horizontally in 500 ml glass jars

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Table 1. In vitro establishment of cultures derived from two mature (100-year-old) oak trees.

Clone Source of N Contamination Reactivity SH+SD L4-SD explants (%) (%) (mm)

SL1 Old branches 75 5 36 1.64-0.2 15.84-1.6 Twigs 36 11 0 0 0 Current season 25 50 0 0 0

SL3 Old branches 50 12 20 2.04-0.5 13.64-1.4 Twigs 49 4 0 0 0 Current season 33 40 0 0 0

Explants were taken from shoots forced in a growth cabinet from crown branch segments (both old branches 3-5 cm in diameter and twigs of last year's growth), and from the current season's growth. Data were recorded after 6 weeks of culture. N, number of initial explants; SH, mean number of shoots longer than 6 mm per reactive explant; L, length of tallest shoot on each reactive explant; SD, standard deviation.

(with glass lids fixed with plastic film) containing 70 ml of multiplication medium, and were transferred to

fresh medium at 2 weeks during the 4-week multipli-

cation cycle. To further increase propagation efficiency, horizon-

tal shoot explants were recycled as follows. Decapitat-

ed shoots, 20 mm long, from five out of the seven established oak clones (see Table 3) were cultured hor- izontally in glass jars. At the end of the 4-week mul- tiplication cycle new shoots longer than 8 mm were

excised for subculture or rooting and the mother shoots

were recultured on fresh medium, again horizontally, to obtain a second crop of shoots. The mother shoots

were recycled four times in all. After 2 and 4 weeks

of each multiplication cycle, the following variables

were determined:

- the in vitro reactivity;

- the number of shoots longer than 8 mm per hori-

zontal explant;

- the number of rootable shoots (longer than 14 mm)

per horizontal explant; and

- the length of the tallest shoot produced by each

explant.

There were eight culture jars per clone with six explants per jar. For each clone the recycling experiment was

performed twice. Rooting experiments were performed for five of

the seven established clones (see Table 4). Shoots 15- 25 mm long were isolated from mother shoot multi- plication cultures. The rooting medium consisted of

GD medium with macronutrients reduced to one third strength and supplemented with 14.8 ~tM of IBA. After

Fig. 2. Shoot development from an explant taken from a branch forced shoot, after 6 weeks of initial culture (Clone San Esteban). Diameter of the test tube = 2.0 cm.

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Table 2. Flushing capacity of branch segments (3-5 cm in diameter) from six oak trees, and, in vitro performance of explants from the flushed shoots.

Clone Hushing in growth cabinet In vitro cultures

(age: in Total Mean length (mm) N Contamination Reactivity SH4-SD L4-SD years) number of (range) (%) (%) (mm)

flushed shoots

Sainza 3 26 38 17a 14 2 .54-1 .5 22.54-17.5 (300) (19-85) 22

63n 56 3.14-2.1 18.64-7.3

Sainza 7 31 30 27a 0 0 0 (300) (19-55) 13

61n 4 1.04-0 6.54-1.5

Monasterio 29 33 28a 11 1.0-4-0 6.74-0.9 (80) (18-55) 1

78n 18 1.54-0.8 13.94-11.1

Tixon 27 64 27a 27 1.04-0 6.0-1-0 (70) (18-156) 3

91n 21 1.44-0.8 7.64-3.1

Pega 49 37 38a 0 0 0 (80) (17-69) 10

140n 0 0 0

San Esteban 47 58 42a 19 2 .84 -1 .1 17.64-6.9 (100) (33-115) 12

210n 51 3.54-1.6 21.3-t-8.2

F1 2 30 16" 6 19 1.34-0.3 10.34-0.4 (100) (30-30)

* Data recorded without distinction between a and n explants Data on initial cultures were recorded after 6 weeks. Sainza 3 and Sainza 7: Branch segments were collected at two levels (3 and 7m from the soil) on the same tree. N, number of initial explants; SH, mean number of shoots longer than 6 mm per reactive explant; L, length of tallest shoot on each explant; SD, standard deviation; a, shoot tip explants; n, nodal explants.

7 days the shoots were transferred to fresh med ium

( 1 / 3 G D ) without auxin. Results were recorded 1 month

after the beginning o f auxin treatment. For each clone,

18-24 shoots were used and the exper iments were

repeated three times. An initial 5-day dark period was

appl ied for some clones.

The significance o f differences among the rooting

results o f different clones was established by one-way analysis o f variance. Percentage data were subjected

to arcsine t ransformation prior to analysis.

Results

Initiation of cultures

After about 8 - 1 0 days in the growth cabinet, shoots

began to emerge through the bark o f the branch seg-

ments. Af te r 15-20 days, v igorous shoots were col-

lected and used as the source o f explants for in vitro establ ishment (Fig. 1). F lushed shoots were normal ly

associated with branch scars, a l though in some cases

Fig.3. Shoot multiplication in clone SL 1 during the second reculture of a horizontal mother shoot. Scale in cm.

shoots developed elsewhere on the segment. Old SL1 branch segments produced an average of five 20-100 mm shoots, and old SL3 branch segments an average of two 40-100 mm shoots.

Attempts to initiate cultures from the previous or the current season's growth were unsuccessful (Table 1). In the latter case, moreover, the contamination rate reached 50%, because the shoots had flushed directly on the tree. However, the explants from shoots flushed from branch segments had reactivities of 36% (SL1) and 20% (SL3).

In view of these results, only the method of section- ing branch segments was applied to six other adult oak trees. Flushing occurred in all six clones, with between two and six 17-156 nm shoots per section (Table 2). To investigate whether topophysical effects affect in vitro performance, branch segments were collected at two levels on the Sainza tree: 3 and 7 m from the soil. Branch segments from the 3 m level flushed after 17 days, as against 28 days for branches from the 7 m level, and explants derived from the lower branches (Sainza 3) performed better in vitro than those from the higher branches (Sainza 7).

In general, nodal explants were more reactive than apical explants and produced more and longer shoots. Of the six clones, the only totally unreactive one was Pega, whereas the most reactive clones were San Este- ban (Fig. 2) and Sainza 3.

Shoot multiplication stage

After several subcultures shoot growth and vigour and multiplication rates declined if the explants were cultured vertically. In clone SL1, for example, the

291

multiplication coefficient peaked at 4.1 in the third subculture, falling thereafter to 1.3 in the eighth. How- ever, when decapitated shoot explants were cultured in a horizontal position, the new shoots that devel- oped from the axillary buds exhibited vigorous growth and higher multiplication rates than the vertical cul- tures. Furthermore, recycling improved the efficien- cy of propagation. In the successive recultures, small shoots of the previous reculture's growth developed to cropping size, and new shoots developed from axil- lary buds remaining around the basal stumps of shoots excised previously (Fig. 3). Table 3 lists, for each of five clones, the results of recycling a single explant. In three of the five clones, the number of shoots per explant peaked in cycle (reculture) 2 and remained rea- sonably high at least until cycle 4. However, the most marked effect was the increase in shoot length (even in those clones yielding most shoots in cycle 1), and hence in the number of shoots suitable for rooting. Fur- thermore, some clones had vigorous, rootable shoots after reculture for only 2 weeks, showing that after the first cycle shoots could be collected for rooting every 2 weeks and the subculture period shortened. Although, in general, the values of all the variables evaluated declined after four culture cycles, the number of recul- tures worth doing varied from two for the Sainza clone to five or six for San Esteban and SL3.

Preliminary rooting experiments

The rooting performance of the clones tested is listed in Table 4. An initial 5-day dark period was necessary for clones Sainza, SL3 and SL1, which without this dark period had rooting rates of only 0, 14 and 5% respectively. Roots (Fig. 4) generally began to emerge during the second or third week after the beginning of rooting treatment, although in clone SL3 they only became visible after day 20 or 22. Clone identity sig- nificantly influenced rooting percentage (p < 0.005) and root length (p < 0.05), but not the number of roots formed (Table 4).

Discussion

The main result of the present study was the estab- lishment and multiplication of in vitro cultures of old oak trees, using explants from shoots flushed from branch segments. This procedure appears to generate at least partially rejuvenated or reinvigorated material able to produce reactive cultures. It must be empha-

292

Table 3. culture.

Effects on productivity of recycling 20mm mother shoots of five mature clones of Quercus robur after 2 and 4 weeks of

Reculture number 2 Weeks 4 Weeks SH>8 mm SH>14 mm L(mm) Reactivity(%) SH>8 mm SH>14 mm L(mm) Reactivity(%)

C l o n e : M o n a s t e r i o

1 3.7 4-0.5 0.24-0.2 10.74-1.1 97 6.14-0.8 2.64-0.5 23.74- 3.5 97

2 3.64-0.4 1.14-0.3 19.74-3.7 97 7.84-1.8 3.54-1.2 27.7 4-2.5 97 3 3.44-0.9 1.34-0.4 20.74-5.1 100 7.14-1.4 2.94-0.8 27. 74-4.1 100

4 3.04-1.2 0.84-0.6 14.04-4.1 90 4.64-1.4 1.84-1.3 20.0 4-6.4 90

Clone:Sa lnza-3

1 3.4 4-0.3 0.24-0.2 10.14-1.6 95 7.04-1.2 1.64-0.6 16.34- 2.6 98 2 4.04-0.9 0.54-0.2 13.24-1.0 95 5.04-1.0 1.34-0.5 16.6 4-1.8 98

3 2.14-0.3 0.24-0.2 9.64-1.4 93 2.94-0.8 0.24-0.1 10.34-1.1 95 4 1.44-0.4 0 7.14-0.5 43 1.54-0.5 0 7.34-1.6 36

Clone:SL3

1 2.94-0.9 1.04-0.2 13.84-1.8 94

2 4.5 4-0.8 1.14-0.3 17.44-2.2 97 8.34-1.7 3.04-0.8 25.44- 2.6 100 3 4.54-1.1 0.84-0.7 14.44-3.9 94 7.74-1.4 2.24-1.2 21.4 4-4.5 100 4 3.84-0.7 0.34-0.4 12.64-3.9 91 7.64-1.2 2.14-0.6 21.3 4-4.9 100

Clone:SL1 1 7.24-1.3 3.04-1.0 1.74-5.7 97

2 3.6 4-0.7 0.94-0.2 19.14-3.1 97 5.04-0.7 2.44-0.7 28.24- 3.3 97 3 2.34-0.2 0.74-0.5 21.94-12.7 82 4.04-0.9 1.24-0.8 22.54-11.6 83 4 1.94-1.1 0.34-0.4 11.84-5.8 49 3.34-2.4 1.14-1.3 18.8 4-10.1 51

Clone: San Esteban

1 2.44-0.4 0. 7+0.4 13.64-1.3 96 5.74-0.5 3.34-0.8 27.44-4.1 100 2 5.34-0.9 2.54-0.8 23.64-6.1 100 7.44-1.0 4.74-1.5 30.64-5.3 100 3 3.54-1.1 1.94-1.0 23.44-6.6 100 6.04-1.5 3.84-1.2 31.14-4.3 100 4 3.04-1.1 1.64-1.0 23.74-8.0 96 4.64-1.3 3.14-1.8 33.0 4-7.7 100

- Data not recorded The mother shoots, placed horizontalty, were transferred to fresh multiplication medium at 4-week intervals after excision of axillary shoots (reculture). SH, mean number of shoots per explant; L, length of tallest shoot per explant (mm). Values are means -4- standard deviation.

sized that there have been no reports of success in

establishing cultures directly from buds of the current

season's growth taken from the crown of mature oak

trees.

Although the flushed shoots seemed generally to

be associated with branch scars, this association was

not always evident. It is therefore, difficult to precisely

state whether some of the induced shoots were or were

not of adventitious origin. Ikemori (1987) considered

that shoots developed in the greenhouse on branch seg-

ments from a 10-year-old Eucalyptus grandis tree were

epicormic. Evers et al. (1990) distinguished between

shoots associated with branch scars and those else-

where oh trunk sections of an 8-year-old oak tree. The

former were called accessory, since they were consid-

ered to have been once associated with the terminal

bud of the branch and to have remained dormant since

then, while the latter were called epicormic since these

emerged from a bud not formed in the last growing

season. We think that, in our study, both accessory

and epicormic shoots developed from preformed buds

induced to flush when isolation of the branch segments

broke apical dominance. Physiologically, epicormic

and accessory shoot formation is difficult to interpret

because of the interrelationships among several envi-

ronmental effects, including the effects of injury, tem-

perature, light, water and nutrients. The most accepted

theory about formation of the different types of buds is

based on an inhibition control factor (probably auxin)

produced in the crown (Bachelard 1969).

293

Table 4. Effect of genotype on rooting ability of five oak clones after 1 month in rooting medium.

Clone Rooting+SD Root number4-SD Root length (mm)+SD (%)

Monasterio 37-t-7.9 1.54-0.3 10.04-3.9

Sainza 3* 164-0.8 1.74-0.5 32.24-4.2

Sainza 7* 0 0 0

SL3* 404-13.1 2.04-0.3 28.34-9.6

SLI* 154-2.3 1.14-0.1 28.04-11.4

San Esteban 464-8.1 2.14-0.2 32.64-4.1

p<0. 005 ns p<0.05

*: Values achieved with an initial 5-day dark period, ns: not significant Rooting was induced by culture in medium supplemented with 14.8 laM IBA for 7 days. Each value represents the mean 4::kstandard deviation (SD) of three repeated experiments with 18 replicates per clone.

Fig. 4. Root development on an oak shoot after one month in rooting medium (Clone San Esteban). Scale in cm.

The forced flushing method described here is relat- ed to the severe pruning method used for rejuvenation purposes (Franclett 1977). The flushed shoots induced on oak old -branch segments showed vigorous growth, with long internodes and leaves resembling a more juvenile looking type (less lobed). It appears that this material was sufficiently rejuvenated or reinvigorated to provide reactive explants. However, the cause of Pega explant failure may be related to its genotype, since there appears to be no clear relationship between flushing capacity and in vitro reactivity. The multi- plication rates of our reinvigorated oak material were similar to those of cultures of juvenile origin (San-Jos6

et al. 1988). Ikemori (1987) concluded that epicormic shoots produced by eucalyptus branch segments were of the juvenile type and that explants could be excised from these that were capable of providing good multi- plication and rooting in vitro. Rejuvenation by forced flushing of trunk sections has been applied to Sequoia sempervirens (Hunter & O'Donnell 1988) and Q. robur (Vermeer et al. 1991), but this technique has the draw- back that the selected tree must be felled.

The decline in the proliferation capacity of ver- tically subcultured explants is difficult to interpret. Juncker & Favre (1989) reported a similar decline in the multiplication factors of oak clones derived

294

from mature material that had previously been graft- ed to induce rejuvenation. Subculture of horizontal decapitated explants seems to reinvigorate oak cultures more than subculture of vertically placed explants. We have observed no decline in shoot multiplication after 2 years of subculturing with horizontal explants, which suggests that these cultures have entered the 'stabilized' phase as defined by McCown & McCown (1987). Horizontal explant orientation also increased shoot production in juvenile Q. robur (San-JosE et al. 1988) and other woody species (Zimmerman & Ford- ham 1989; McClelland & Smith 1990), and was essen- tial for in vitro shoot proliferation of Q. rubra (Vieitez et al. 1993). Its action in promoting axillary shoots is believed to result from altered distribution of growth regulators and greater uptake of medium constituents (Mackay & Kitto 1988). It is known that the produc- tion of ethylene increases under mechanical stress such as that caused by horizontal orientation (Leopold et al. 1972). The availability of ethylene, regulated endoge- nously, is essential to the released bud on a decapitated plant in order to sustain its subsequent development into a lateral shoot, possibly acting by reducing auxin transport (Yeang & Hillman 1984). Increased ethylene production would explain the increase in axillary shoot development and growth in the horizontal cultures.

In the present study, reculturing improved the per- formance of cultures from adult crown material that had previously been reinvigorated by forced flushing. Often rejuvenation is achieved by sequential treat- ments. In vivo rejuvenation methods are sometimes used before in vitro ones (Pierik 1990). The in vitro manipulation of subculture explants needs to be opti- mized.

The rejuvenation achieved in this study cannot be regarded as complete, because rooting ability was not restored to the same level as in juvenile material (Vieit- ez et al. 1985; Juncker & Favre 1989). Genotypic differences may play a role, not only in initial reactiv- ity but also in rooting capacity. Chalupa (1988) found great differences in rooting percentages (between 14 and 86%) of micropropagated shoots of 12 adult oak clones, even though the original material was taken from stump sprouts, epicormic shoots and shoots from the lower branches of the crown and was therefore more juvenile than our crown material. Rooting rates of 80-90% have been reported for cultures from rejuve- nated epicormic shoots flushed from branch segments of eucalyptus (Ikemori 1987) and oak (Evers et al. 1990), though in these cases the mother trees were only 10 and 8 years old respectively. This age dif-

ference may account for our inferior rooting rates. The efficiency of rejuvenation decreases with age; the older the plant, the stronger the treatment needed to achieve rejuvenation (Franclet 1980). Furthermore, the shoots used by Evers et al. (1990) were induced on trunk sec- tions, and this topophysical difference may also have favoured their rooting rates. In principle, the basal and proximal parts of a tree may retain more juvenile characteristics (Bonga 1987). This could also explain the differences in rooting between the Sainza cultures derived from two positions on the tree.

Acknowledgements

This research was partially supported by the Com- mission of the European Communities through the ECLAIR programme. Contract AGRE 0067. Thanks are given to Dr. J.M. Bonga (Forestry Canada, Freder- icton, Canada) for critical reading of the manuscript. J.B.A.M. is indebted to the Consellerfa de Cultura, Educaci6 i Ciencia, Generalitat Valenciana, for a fel- lowship.

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