Journal
Journal of Applied Horticulture, 19(3), 215-217, 2017
Appl
Comparing micro-cutting and mini-cutting technique for the
clonal propagation of Metrosideros excelsa
L. Sabatino*, F. D’Anna, R. Nicolosi and G. Iapichino
Department of Agricultural, Food and Forest Sciences, University of Palermo, Viale delle Scienze, Palermo, Italy.
*E-mails: leo.sabatino@unipa.it
Abstract
Metrosideros excelsa is an erect evergreen branched shrub native to the North Island of New Zealand which is grown in private and
public gardens for its foliage and attractive red flowers. M. excelsa plants are commercially propagated by soft and semi-hard cuttings.
Therefore, new and more reliable propagation techniques derived both from conventional and advanced propagation methods should
be considered to support Metrosideros nursery development. The mini-cutting and micro-cutting techniques were compared for the
clonal propagation of M. excelsa. Ex vitro rooted micro-cuttings were superior to mini-cuttings in terms of rooting percentage, root
number per cutting and root length by 23.5, 42.5 and 2.4%, respectively. However, the mini-cutting technique can be considered an
alternative reliable propagation method because it is less expensive than the micro-cutting technique and because it does not depend
on tissue culture to supply stock plants.
Key words: Vegetative propagation, micropropagation, stock-plant, rooting, Pohutukawa
Introduction
Metrosideros excelsa, commonly known as Pohutukawa, is an
erect evergreen branched shrub native to the North Island of New
Zealand. Grown in private and public gardens for its foliage and
attractive red flowers the species prospers in the coastal areas
of the Mediterranean region, particularly in the mild climate
of southern Italy and Sicily where it is also used in edges or in
large pots and tubs. Metrosideros propagation by seed is feasible,
however, since many plants of the landscape are normally hybrids
or cultivars, cutting is the the most adopted propagation method.
Plants are commercially propagated by soft and semi-ripe cuttings
from spring to fall (Bryant, 2003). However, according to Bryant
(2003). M. excelsa percent rooting can vary from 40 to 80%
depending on cultivar variations in the ability to root. Therefore,
new and more reliable propagation techniques eventually derived
both from conventional and advanced propagation methods are
an urgent need among scientists, growers and extension specialist
in order to support plant nursery management. Micropropagation
is an alternative multiplication method, especially to rapidly
increase M. excelsa new selections (Iapichino and Airò, 2008;
Clemens et al., 1999). Therefore, ex vitro rooting of in vitro
multiplied micro-cuttings, as described by Iapichino and Airò,
(2008) reduces plant tissue culture costs and may improve the
efficiency of commercial laboratories works involved in M.
excelsa micropropagation production. However, from other side
the efficiency of production may depend on tissue culture to
supply stock plants.
The mini-cutting technique is similar to the micro-cutting one, as
mini-shoots from plants propagated by conventional cutting are
used as source of propagation material (Chinnaraj and Malimuthu,
2011). However, since the former method does not rely on the
micropropagation stage, can be a less costly alternative. The
mini-cutting technique has expanded rapidly in the forestry sector,
for example, to clonally propagate Eucaliptus species (Xavier
and Wendling, 1998; Assis, 2011; Bindimadhava et al., 2011).
Nevertheless few published data is available concerning the effect
of mini-cutting technique on the performance of ornamental
plants, such as Acer palmatum and Paulownia fortunei (Stuepp
et al., 2015; Stuepp et al., 2016).
Based on the above considerations, the aim of this study was to
provide new knowledge on this topic by comparing the microcutting and mini-cutting technique for the clonal propagation of
M. excelsa.
Materials and methods
The research was conducted at the Department of Agricultural,
Food and Forest Sciences (SAAF) of the University of Palermo,
in the northern coast of Sicily (Italy) (long. 13° 19’ E, lat. 38°
9’ N). A ten year old mother plants of M. excelsa was subjected
to severe pruning in June 2016. Juvenile shoot tips, 2-3 cm in
length arisen in late winter 2017 from the dormant buds of the
stump (Fig. 1a) were harvested and used as mini-cuttings in the
rooting experiment.
In vitro shoot cultures of M. excelsa were established as described
by Iapichino and Airò (2008) from primary explants collected
from the above mentioned mother plant (Fig. 1b). After five
weeks of culture, axillary shoots (@ 20 mm long) developed on
primary explants were excised, the terminal buds removed and
the resulted stems cut into segments, bearing one node. Nodes
were subcultured every four weeks in the multiplication medium
(Iapichino and Airò, 2008) to increase the stock of shoot cultures.
Shoots were then subcultured in the same medium without growth
regulators for 2 more weeks. Randomly selected individual
shoots ≈ 2.5 cm long with intact apices and 3 to 4 leaves were
cut from stock culture and used as micro-cuttings in the rooting
experiment.
Both mini-cuttings and micro-cuttings used in the propagation
Journal of Applied Horticulture (www.horticultureresearch.net)
216
Micro-cutting and mini-cutting technique for the clonal propagation of Metrosideros excelsa
ANOVA analysis. Mean separation was
performed by Duncan Multiple Range
Test. All the statistical analyses were
performed using SPSS software version
14.0 (StatSoft, Inc., Chicago, USA).
Results and discussion
The present results demonstrated that
the cutting survival accounted for 97.3%
and 95% in micro-cuttings and in minicuttings, respectively. No significant
effect of the cutting method was observed
for cutting survival (Table 1).
Ninety-six percent rooting was observed
after three weeks from planting for
plants propagated by the micro-cutting
technique. Rooting dropped from 98.3 to
55% adopting the mini-cutting technique
(Table 1). Five weeks after cutting
insertion in the rooting media, rooting
percentage accounted for 98 and 75 %
in micro-cuttings and in mini-cuttings,
Fig. 1. a) Mini-cutting mother plant of M. excelsa. b) Micro-cutting mother plant of M. excelsa. c) respectively (Table 1). Moreover, after 3
Rooted mini-cutting (left) and micro-cutting (right) of M. excelsa. d) Acclimatized mini-cutting (left) weeks from planting, root number formed
and micro-cuttings (right) of M. excelsa.
experiment were trinodal with the lower cut below a node at the
base of each cutting. All cuttings were planted in plastic trays
containing a peat-perlite mixture 1:1 (v/v). Propagation was
performed, in March 4, 2017 in an unheated greenhouse covered
with clear polyethylene (PE) and external 70 % shade cloth. Air
temperature in the greenhouse was 12-14 °C during the night and
16-20 °C during the day. To verify the mini and micro-cutting
rooting response trays were placed on a bottom heated bench at
constant temperature of (22 ± 2 °C). The medium was watered and
the trays covered with clear polyethylene (PE) to maintain cutting
turgidity. Ventilation of the plantlets was increased with time by
increasing size of the holes made in the plastic, and after three
weeks the plastic was removed. After 6 weeks, acclimatized single
plants were transferred to plastic pots (diameter 9 cm, height 10
cm) containing the same growing mixture and grown under the
greenhouse conditions as described previously.
The rooting experiment was set up in a randomised complete
block design consisting of two propagation techniques (minicutting and micro-cutting) with three blocks per treatment and
10 shoots per block giving total of 30 shoots. After three and
five weeks, data were recorded as percentage of cutting survival,
percentage of shoots rooted, number of roots per shoot and mean
root length. In addition, plant height at root collar after five weeks
and after acclimatization (six weeks from planting) was recorded.
Percentage data were subjected to arcsin transformation before
Table 1. Effects of the propagation technique on M. excelsa micro- and
mini-cutting survival and rooting percentage
Propagation
technique
Cutting survival Rooting after
(%)
3 weeks (%)
Rooting after
5 weeks (%)
Micro-cutting
97.3n.s.
96.3a
98.0a
Mini-cutting
95.0
55.0b
75.0b
In each column, means followed by the same letters are not significantly
different at the P ≤ 0.05 level by Duncan’s Multiple Range test.
through the micro-cutting technique was significantly superior
by 18.7 % compared to the mini-cutting technique (5.7 and 4.8
roots per cutting, respectively) (Fig.1c). Data collected after five
weeks followed a similar trend (12.7 and 7.3 roots per cutting,
in micro-cuttings and in mini-cuttings, respectively) (Table 2).
The average root length for cutting after 3 weeks from planting
were 5.1 cm for micro-cuttings and significantly inferior for
mini-cuttings (4.2 cm) (Table 2). Furthermore, root length after
5 weeks from planting was not significantly affected by the
propagation techniques tested. In both types of rooted cuttings,
upper shoot growth was manifested in new leaf expansion and
stem elongation. After five weeks from planting, plant height at
the root collar measured 4.9 and 4.7 cm for micro-cutting and
mini-cutting derived plants, respectively (Table 2). After six
weeks, acclimatized plants did not significantly differ in terms of
plant height (Table 2; Fig. 1d). Micro-cutting and mini-cutting
Table 2. Effects of the propagation technique on the number of roots per cutting, root length and plant height of M. excelsa rooted micro- and minicuttings
Type of
technique
Micro-cutting
Roots per cutting
after 3 weeks (No.)
5.7a
Roots per cutting
after 5 weeks (No.)
12.7a
Root length after 3
weeks (cm)
5.1a
Root length after 5
weeks (cm)
Plant height after 5
weeks (cm)a
Plant height after 6
weeks (cm)b
29.6n.s.
4.9n.s.
5.1n.s.
Mini-cutting
4.8b
7.3b
4.2b
28.9
4.7
In each column, means followed by the same letters are not significantly different at the P ≤ 0.05 level by Duncan’s test.
a
End of rooting. bEnd of acclimatization.
Journal of Applied Horticulture (www.horticultureresearch.net)
4.9
Micro-cutting and mini-cutting technique for the clonal propagation of Metrosideros excelsa
rooted plantlets were successfully established in soil and are under
evaluation for their clonal fidelity and agronomic behaviour.
It is well established that stem cutting propagation is the most
dominant method of asexual propagation for the production
of many ornamental shrubs (Sabatino et al., 2014; Kashefi
et al., 2014; Kaviani and Gholami, 2016; Pacholczak, 2015;
Zhang et al., 2015). According to Sicilian nurseries involved
in conventional propagation of M. excelsa, rooting success can
vary depending on cutting harvest period, type of cutting and
rooting conditions. However, to reach an acceptable rooting rate,
conditioned propagation greenhouses supplied with bottom heat
benches and a mist or fog air humidification system are required.
In the current experiment, with a less sophisticated equipment,
we were able to obtain, after five weeks, a 98 and 75 % rooting
success with micro and mini-cuttings, respectively.
The rooting superiority of mini and micro-cuttings has been
related to the higher regenerative capacity in the two groups of
juvenile propagules as compared to conventional stem cuttings
(Assis et al., 2004). Our results are also consistent with those of
Iapichino and Airò (2008) who found 90 % rooting in M. excelsa
micro-shoots after 4 weeks exposure to bottom heated bench.
Our results also demonstrated that micro-cuttings rooted more
rapidly and efficiently than mini-cuttings. Our findings are
partially in line with those of Titon et al. (2006) who reported
Eucalyptus grandis micro-cuttings having a higher rooting
rates than mini-cuttings but only for difficult-to-root clones.
On this respect, we must point out that in our system we rooted
micro-cuttings which were directly collected from in vitro stock
cultures and mini-cuttings which were directly harvested from a
severely pruned mother plant. On the contrary, in the experiment
of Titon et al. (2006), mini-cuttings were collected from miniclonal edges (mini-stumps) obtained by rooted mini-cuttings
derived from rooting conventional stem cuttings and “micro
cuttings” from micro-clonal edges obtained in turn by rooting
micro-shoots from in vitro cultures. Indeed, Assis et al. (2004)
suggest that mini-cuttings may require a sequence of propagation
cycles (serial propagation) to acquire a full potential rooting
capacity. Therefore, our results could be attributed to the fact
that, differently from mini-cutting, micro-cuttings had been
subjected to in vitro propagation cycles and consequently might
have reached propagation cycles and with superior potential
rooting capacity. However, although rooting performance on
mini-cuttings resulted inferior to that of micro-cutting in terms
of rooting percentage, root number per cutting and root length,
the mini-cutting technique can also be considered a reliable M.
excelsa propagation method because it is less expensive than
micro-cutting technique and because it does not depend on tissue
culture to supply stock plants.
217
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Received: April, 2017; Revised: May, 2017; Accepted: July, 2017
Journal of Applied Horticulture (www.horticultureresearch.net)