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In vitro studies on Eucalyptus globulus rooting ability

Article  in  In Vitro Cellular & Developmental Biology - Plant · January 1997

DOI: 10.1007/s11627-997-0032-8

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In VitroCell.Dev.Biol.--Plant33:1-5,January1997
© 1997Societyfor In VitroBiology
1071-2690/97 $05.00+ 0.00

IN VITRO STUDIES ON EUCALYPTUS GLOBULUS ROOTING ABILITY

H. TRINDADE ANDM. S. PAIS

Soporcel ForestryResearch Centre, P.O. Box 15, 2065 Alcoentre, Portugal (H. T.); Centerfor Plant Biotechnology,
Department of Plant Biology, Faculty of Sciences,
Universityof Lisboa, Bloco C2, Campo Grande, 1700 Lisboa, Portugal (M. S. P.)

(Received 30 January 1996; accepted 5 August 1996; editor G. C. Phillips)

SUMMARY
Micropropagation has the potential to quickly introduce selected genotypes of adult Eucalyptus globulus clones and it is
now widely used in Portugal as a part of genetic improvement programs. Several clones have been established and multiplied
in vitro. The different clones have individual requirements for successful rooting. Rejuvenation was achieved at different
periods after culture initiation for the different clones. Subculturing preceding rooting in multiplication medium supple-
mented with riboflavin and cholene chloride allowed the increase of rooting ability for several clones tested. Removal of
boron from the rooting medium increased rooting by 10%. Indolebutyric acid (IBA) dipping before transfer to the rooting
medium resulted in a rooting percentage of 80-95% for the best clones tested. Acclimatization was performed without
difficulties (90-95% success) and the rooted plants were either planted directly or used as mother plants for further cutting
production, depending on the needs. The results described in this paper increase the commercial feasibility of the micro-
propagation system for E. globulus.
Key words: rooting; boron; rejuvenation; Eucalyptus globulus.

INTRODUCTION
Eucalyptus globulus is a very important tree for the pulp and paper
industry. It is an exotic species, a native from Australia that was
introduced in Portugal in the 19th century. Today, E. globulus is the
main eucalypt species planted in Portugal and presently it occupies
an area of 530 000 ha. Eucalyptus pulps are preferred due to their
lower production cost and their excellent bulk, softness, flexibility,
formation, opacity, and porosity, which make them particularly suit-
able for tissue, printing, and writing grades of paper (Sidaway, 1988).
In Portugal, the poor genetic quality of most stands was responsible,
until some years ago, for the low production of wood per hectare. For
this reason, the present genetic improvement program has been im-
plemented. Biotechnology plays an important role in this program
because adult trees are difficult to propagate through conventional
techniques such as cuttings. Although cuttings are used to produce
most of the plants used today in clonal plantations, for some of the
best performing clones it is difficult to achieve a good rooting ability.
To overcome this problem, we developed a micropropagation system,
based on in vitro propagation through axillary bud proliferation. It
allows the successful propagation of adult recalcitrant trees (10-30
yr old). The plantlets can be planted directly in the field for clonal
tests or can be used as mother plants to be propagated through cut-
tings. Micropropagation of Eucalyptus species has been reported by
some authors. Le Roux and Van Staden (1991) provided information
on tissue culture of 30 different Eucalyptus species. In most cases,
the explants used were obtained from seedling-derived plants or from
juvenile materials. When explants were obtained from adult trees,
the plantlets obtained then presented very poor rooting ability. Ben-
nett et al. (1994) reported a procedure for micropropagation of 5-yr-
old E. globulus trees using coppice stumps. In this paper, we report
on a micropropagation system using the epicormics obtained from
the branches of adult E. globulus trees, which enables the successful
micropropagation and rooting of different clones from this species.
MATERIALSAND METHODS
Clonal lines from different trees were established as follows: Epicormic
shoots obtained from branches of adult Eucalyptus globulus trees (10~30 yr
old) were surface sterilized with 5% calcium hypochlorite for 5-10 min,
washed repeatedly with sterilized water, and cultured on a modified de Fos-
sard (1974) medium supplemented with N6-benzyladenine (BA) 1.11 or 2.22
gM and indole-3-butyric acid (IBA) 0.1 or 0.49 pA/, as described in Trindade
et al. (1990). The use of kinetin, alone or in combination with IBA, was tested
in the initial experiments. Later on, the cultures obtained on media containing
these growth regulators were discarded due to reddening of the shoots. Glu-
cose (30 g/l) was used as carbon source. The effect of different composition
of de Fossard (1974) multiplication media, preceding rooting (FM0, FM1,
FM2, and FM3), on further rooting ability was tested (Table 1). Every 3-4
wk, the shoot clusters obtained were sectioned and small groups of shoots
were transferred into new culture medium. Cultures were grown in a culture
room with a temperature of 22 + 2° C and a photoperiod of 16 h light and
8 h darkness provided by Philips 83 fluorescent lamps. Light intensity was
50 ttE m 2s-1.
Rooting ability was assayed for each clone at different periods after culture
establishment. This was performed by dipping the base of the individual
shoots in an IBA solution (0.49, 1.22, or 2.45 mM concentration) for 5-30
rain. The shoots were then transferred to a basal de Fossard (1974) medium,
modified as in Badia N'Kanda (1981) without growth regulators. Phloroglu-
cinol 39.65 ttM was added to the cooled autoclaved culture medium by filter
sterilization (James and Thurbon, 1981). In the rooting phase, glucose con-
centration was lowered to 20 g/l. In a separate experiment, rooting ability was
assayed in the presence or absence of boron (50.13 ~M) in the rooting me-
dium. The shoots were kept for 4 d in darkness following IBA dipping, and
then transferred to the culture room under light conditions.
2 TRINDADE AND PAlS

TABLE 1 then transferred to the greenhouse. Slow release fertilizer (Osmocote 11:22:9
+ 6 Mg) (Sierra) was incorporated in the soil mixture at a rate of 5 g/1. The
MODIFICATIONS OF DE FOSSARD (1974) MEDIA TESTED AS fungicide Previcure (AgrEvo), at a concentration of 1 mr/l, was also used to
MULTIPLICATION MEDIA DURING SUBCULTURES PRECEDING irrigate the small pots 1 d before acclimation in order to prevent fungal
ROOTING OF ~UCALYPTUSGLOBULUS,AND SUBSEQUENT diseases. When the plants were about 8-10 cm high, they were hardened
ROOTING FREQUENCIES. VALUES OF ADDITIVES ARE outside before being planted in the field.
CONCENTRATIONS EXPRESSED IN IJM. THE VALUES ARE
STATISTICALLY SIGNIFICANT (P = 0.0003). RESULTS
Choline Rooting Ability
IBA° BA Riboflavin Chloride (mean % +-- SE) The multiplication conditions of E. globulus have been previously
described (Trindade et al., 1990) and were not clone dependent.
FMO 0.1 1.11 -- -- 60 + 2 However, each clone had a particular phenotype in the multiplication
FM1 4.92 1.33 7.97 10.03 85 + 6 medium that was used to identify them in vitro. In the rooting phase,
FM2 0.1 1.11 7.97 10.03 80 + 5
FM3 0.49 1.11 7.97 10.03 85 + 2 each clone had a defined root architecture, regardless of the rooting
ability. Some individuals, coming from different clones, had one main
IBA = indole-3-butyric acid; BA = N6-benzyladenine. root (Fig. 1 A), while others had two to three roots (Fig. 1 B) with
several secondary roots.
Although a general protocol could be established for some E. glob-
Rooting ability was assessed 1 mo. after root induction. All the rooting ulus clones, rooting ability was highly clone dependent and the root-
experiments had at least 10 replicates (10 culture vessels with 8 explants ing conditions had to be adapted for some recalcitrant clones. The
each) and each experiment was repeated at least three times. Statistical anal- period of rejuvenation, by in vitro subculture, necessary for rooting
ysis was performed for all experiments using analysis of variance (ANOVA). to proceed with good rates varied from clone to clone, even when
The results of statistical analysis were considered significant at P < 0.05.
The rooted plantlets were potted in a defined substrate composed of peat and considering clones of the same age and initiated in vitro at the same
sand (50:50--vol/vol), acclimatized for 2 d at 80-95% relative humidity, and time (Fig. 2). The clones AR24, MN144, and RE74 were established

-~-+-~lX) ,

iI

!! ~:~ :~

/ ] l+i • i •.

r
I

A
FiG. 1. Rooted Eucalyptus globulus plantlets after 4 wk on rooting medium. A, Typical rooted plant of SN6, with one main root; B,
typical rooted plant of AR24, with two main roots.
 ROOTING OF EUCALYPTUS GLOBULUS 3

cess. Shoot morphology and rooting ability were similar for plantlets
,oo multiplied on both FM2 and FM3 culture media (Table 1). Both
90 media promoted shoot elongation, which made mechanical separation
8O of the plantlets easier. The best success obtained during the rooting
7O phase was obtained from shoots subcuhured for multiplication on
FM 1 and FM3 culture media. These two media had the highest auxin
~ so contents, which was probably responsible for higher percentage of
~ 4o root induction.
Rooting ability was increased by about 10% in media devoid of
20 boron, in comparison to results obtained on rooting medium supple-
1(1 mented with 50.13 ~tM boron (Fig. 3). These differences were similar
0 -- for all the rooting experiments performed.
5 8 9 10 11 12 13 For most of the clones tested, the best rooting percentages were
Time elapsed since establishment of the clonal line (in obtained when a 10-15 rain dipping was done in a 2.45 mM IBA
months) solution. For some clones, the dipping time had to be increased to
FIG. 2. Rootingability in three clones of Eucalyptus globulus following 25 min, while for other clones IBA 1.22 mM was the best concen-
the rejuvenationperiod. Rootingability is increasing with time but at different tration for 10-15 min dipping time (results not shown). Under the
rates for each clone. The difference between the values presented is statis- best conditions for each clone, rooting abilities varied between 80-
tically highly significant (P = 0.0001). 95% (Fig. 4). Acclimatization was achieved with 90-95% success
70 -,- and, after 3-4, wk the root system was observed coming out from the
68 pot (Fig. 5).
66
DISCUSSION
•- 64
62 Methods for micropropagation of E. gtobulus have been described
=~ 60 by some authors (Mascarenhas et al., 1982; Oka et al., 1982) with
moderate success in regard to rooting. Major improvements have
s8
Iv 5 6
been achieved through the Use of a multiplication medium that acts
as a conditioning medium prior to the rooting phase (Bennett et al.,
54 1994). According to these authors, alternate subculture of shoots on
Bo- Bo+
Culture medium
FIG. 3. Comparison of the rooting ability of Eucalyptus globulus shoots
in a modified de Fossard (1974) medium with and without boron.

at the same time, and for each of them rooting was assayed at different
periods after culture initiation. For AR24, 16% rooting was achieved
after 5 mo. subcuhuring on multiplication media. Rooting ability
assayed after 9 mo. subcuhuring increased to 73%. After 1 yr of in
vitro culture, rooting ability was 95% (Fig. 2). For RE74 and MN144
clones, rooting only started at the 9 'h and 10'h mo. of subcuhuring on
multiplication medium and only low rooting percentages (around
10%) were obtained. Rooting induction frequency on material sub-
cultured for 1 yr was 40% for RE74 clone and 70% for MN144 clone.
Rooting ability was affected by the composition of the multipli-
cation medium preceding rooting (Table 1). The best rooting ability
(80-85%) was achieved for plantlets cultured on multiplication me-
dia with added riboflavin and choline chloride (FM1, FM2, or FM3),
during 1 to 2 mo. preceding rooting. Rooting ability obtained from
shoots subcuhured on FMO multiplication medium, which contained
neither riboflavin nor choline chloride, was only 60%; in comparison
to 80% rooting obtained on material subcuhured for multiplication
on FM2 medium, which contains the same growth regulators as FMO
plus riboflavin and choline chloride. Auxin concentration during
multiplication had an effect on rooting ability. Cultivating the shoots
on culture media with an IBA concentration of 0.49 p~M (FM3) al-
lowed 85% rooting ability, while shoots multiplied on culture me- FIG. 4. Rootedplantlets of Eucalyptus globulus at the time of transferring
dium supplemented with 0.1 p.M IBA (FM2) rooted with 80% suc- to acclimatization conditions.
4 TRINDADE AND PAIS
Ffg. 5. Acclimatizedplant of Eucalyptus globulus in the greenhouse.
Roots can be seen coming out of the pot.
multiplication medium containing either BA or kinetin, improved
shoot growth and multiplication in comparison to shoots grown con-
tinuously on medium containing only one of those cytokinins. In vitro
micropropagation on media containing BA provided less than 25%
rooting, while alternation of the two cytokinins improved rooting up
to 75% when kinetin was used in the last multiplication medium
(Bennett et al., 1994). Root induction was higher for shoots produced
on multiplication medium containing kinetin, when compared to root
induction on shoots formed on multiplication medium containing BA.
In this last situation, shoots formed few roots and often died (Bennett
et al., 1994).
In the micropropagation system described in this paper, BA was
the only cytokinin used. The use of kinetin induced reddening of the
explants and, after the third or fourth subculture, the multiplication
rates decreased leading to poor success (Trindade et al., 1990). The
addition of choline chloride plus riboflavin to the multiplication me-
dium improved subsequent rooting percentages ofE. globulus shoots.
These results agree with those described by Badia N'Kanda (1981),
who obtained good success on Eucalyptus rudis micropropagation
through supplementation of culture medium with these vitamins. The
high auxin/cytokinin ratio in the multiplication medium enabled the
elongation of the shoots, which is very useful for further rooting suc-
cess. Shoot multiplication for one to two periods on enriched multi-
plication medium enhanced rooting to 80-95%. These high rooting
percentages are compatible with an industrial production system. To
the best of our knowledge, such rooting percentages have never been
reported for Eucalyptus globulus adult tree-derived micropropagated
plants.
Removal of boron from the rooting medium improved rooting re-
sponse for all the clones tested, when compared to rooting abilities
on rooting medium with boron. It is normally assumed that boron is
an essential micronutrient for root development. However, the role
of this micronutrient on the development of root primordia is not fully
understood. According to Jarvis et al. (1983), boron can regulate
endogenous auxin levels during root development. In Phaseolus au-
reus stem cuttings, an external supply of boron is essential for ad-
ventitious root development (All and Jarvis, 1988). According to
Robertson and Loughman (1974), some of the effects of boron defi-
ciency may be due to increased levels of endogenous hormones. Ac-
cording to Moncousin (1991), the role of boron is very complex as it
is implicated in the transport of carbohydrates, as well as in the
metabolism of auxins and phenols. To the best of our knowledge,
there is no report concerning the role of boron on Eucalyptus root
formation. According to our experiments, boron had an inhibitory
effect on adventitious root formation.
Rooting ability of E. globulus shoots increased through subcuhur-
ing due to the induced rejuvenation of the explants, as reported by
other authors (Franclet et al., 1987). However, the period of time that
each clone needed for rejuvenation to occur was variable. For clone
AR24, rejuvenation was achieved 6---8 mo. after culture establish-
ment, which can be considered very good for E. globulus. After this
stage was achieved, it was maintained through continuous subcul-
turing. The rejuvenating effect may be due to the BA in the culture
medium. As reported by Franclet et al. (1987), the number of sub-
cultures on a BA-rich medium increases the rejuvenation effect.
Rooting was performed on 0.49, 1.22, or 2.45 mM IBA solution
for different periods of dipping. This was highly clone dependent,
and we did not find a single protocol suitable for all the clones tested.
So, for each new clone established, it may be necessary to adapt the
rooting conditions.
The protocol described here can be routinely used for mass prop-
agation of adult E. globulus clones, although some adaptations may
eventually be necessary to achieve the best rooting percentage of
specific clones.
REFERENCES
All, A. H. N.; Jarvis, B. C. Effects of auxin and boron on nucleic acid me-
tabolism and cell division during adventitious root regeneration. New
Phytol. 108:383-391; 1988.
Badia N'Kanda, A. Eucalyptus rudis Endl. Techniques de micropropagation
par la culture de noeuds in vitro. In: Colloque intemationat sur la
culture in vitro d'essences forestieres. France: AFOCEL; 1981:135-
142.
Bennett, I. J.; McComb,J. A.; Tonkin, C. M., et al. Alternating cytokinins in
multiplication media stimulates in vitro shoot growth and rooting of
Eucalyptus globulus Labill. Ann. Bot. 74:53-58; 1994.
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 ROOTING OF EUCALYPTUS GLOBULUS
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Oka, S.; Yeung, E. C.; Thorpe, T. A. Shoot formation in Eucalyptus globulus
hypocotyl explants. N.Z.J. For. Sci. 12:501-509; 1982.
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