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The effect of rooting media, plant growth regulators and clone on rooting
potential of honeybush (Cyclopia subternata) stem cuttings at different
planting dates
G.S. Mabizela
a,b,
⁎,M.M.Slabbert
b
,C.Bester
a
a
ARC Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch 7599, South Africa
b
Department Horticulture, Tshwane University of Technology, Private Bag X680, Pretoria 0001, South Africa
abstractarticle info
Available online 5 March 2016
Edited by J. van Staden
Cyclopia spp. are shrubsindigenous to the Eastern and Western Cape provinces of South Africa that are used for a
herbal infusion known as honeybush tea. Currently, a large amount of honeybush is still wild harvested; this
imposesa risk of biodiversityloss, shortageof plant materialand extinction of natural resources due tounsustain-
able harvesting. Therefore, in order to improve the plant material and relieve pressure on wild populations, a
study aimed at optimising propagation and adventitious rooting techniques of honeybush stem cuttings was
conducted. In this study, four clones of Cyclopia subternata were set in three different rooting media, with
three concentrationsof IBA growth regulators at six planting dates according to a randomised completeblock de-
sign, to determine the effect on rooting. The data recorded were analysed using an analysis of variance (ANOVA).
This study showed significant differences in the rooting potential of the four clones tested. The highest rooting
and survival percentage, root number and root length were experienced when cuttings were propagated in
rooting media Bark mix and 3mix, treated with growth regulators Seradix® B2 and Seradix® B3. Cuttings
grown in rooting medium Peat mix and with growth regulator Dip & Root™resulted in the lowest rooting and
survival percentage. The ideal time to set cuttings was during March and November with clones SGD7 and
SGD9 producing the highest rooting and survival percentages. The lowest rooting percentage was recorded dur-
ing February. Clone selections SGD1 andSGD7 had the highest rooting success followed by SGD9, andclone SGD6
had the lowest rooting percentage. Cyclopiaspp. may be successfully propagated vegetatively by stem cuttings at
different planting dates in order to improve plant material and quality of honeybush.
© 2016 SAAB. Published by Elsevier B.V. All rights reserved.
Keywords:
Honeybush
Rooting
Vegetative propagation
1. Introduction
Honeybush (Cyclopia spp.) is a shrub of the legume family Fabaceae
and is endemic to the fynbos biome, growing along the coastal
and mountainous regions of the Eastern and Western Cape provinces
of South Africa. Aerial parts of Cyclopia spp. (mainly C. subternata,
C. genistoides and C. intermedia) are used to make honeybush tea,
which is characterised by a sweet, honey-like aroma and distinctive
brown colour (Joubert et al. 2011; Theron et al. 2014). The tea has
been reported to contain low levels of tannins, no caffeine and to have
health-promotingproperties (Joubertetal.,2008). Because of the health
benefits, demand for honeybush tea, both locally and internationally,
has increased worldwide (Joubertetal.2011), necessitating the cultiva-
tion of honeybush as a commercial crop (Spriggs and Dakora 2009).
Harvesting honeybush from the wild in South Africa has shown a
significant increase in recent years, which poses a threat to natural
populations (Mbangcolo 2008; Joubert et al. 2011). A major aim of the
honeybush research programme of the Agricultural Research Council
is to propagate and distribute improved genetic material, especially
higher yielding material (Bester 2013).
The majority of Cyclopia species can be propagated by seed and
vegetatively by stem cuttings (Bester 2013; Mbangcolo et al. 2013).
Seed propagation does not ensure the preservation of selected genetic
characters (Hartmann et al. 2002) whereas vegetative propagation
results in plants genetically identical to the original parent plant and
thus are more uniform (Araya 2005). Vegetative propagation is the
preferred method to supply the expanding industry and the demand
for more uniform product (Spriggs and Dakora 2007; Bester 2013).
Rooting success in stem cuttings of Cyclopia spp. depends on a
number of factors including the season during which the cuttings are
collected, age of thesource plant, cutting size, type of rooting substrate,
irrigation, temperature, rooting growth regulator and the clone
(Hartmann et al. 2002; Soundy et al. 2008). Plant growth regulators
(PGRs) have been successfully employed in many plant species to im-
prove the rootability of stem cuttings (Soundy et al. 2008; Singh et al.
2011a; Saǧlam et al. 2014). These include indole-3-acetic acid (IAA),
South African Journal of Botany 110 (2017) 75–79
⁎Corresponding author at: ARC Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch
7599, South Africa.
E-mail address: mabizelag@ar c.agric.za (G.S. Mabizela).
http://dx.doi.org/10.1016/j.sajb.2016.02.200
0254-6299/© 2016 SAAB. Published by Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
South African Journal of Botany
journal homepage: www.elsevier.com/locate/sajb
naphthalene acetic acid (NAA) and indole-3-butyric acid (IBA) (Adekola
and Akpan 2012; Sardoei et al. 2013). Another factor which creates a
suitable environment for rooting of stem cuttings is the rooting medium
(Tchinda et al. 2013). Adugna et al. (2015) reported the highest rooting
percentage of Vanilla planifolia stem cuttings grown in fine sand rooting
medium (99.3%), followed by cuttings grown in forest soil rooting me-
dium (93.33%). There may also be large differences in rooting ability
among clones of many plant species and with different types of cuttings
(Baltunis and Brawner 2010; McIvor et al. 2014). Various studies have
reported variations in rooting ability within genotypes in the same
locations due to natural genetic variation (Husen and Pal 2003;
Rambaran 2013).
Seasonal timing, the period of the year in which cuttings are taken,
can also effect the rooting of stem cuttings. Ayoub and Qrunfleh
(2006) reported an increase in rooting percentage of olive cultivar
“Nibali”during September (55.6%), while cultivar “Rassei”had the
highest rooting percentage during February (92.6). Studies on the effect
of season on rooting of stem cuttings have also been conducted in other
plant species (Bushal et al. 2001; Agbo and Obi 2008; Khosla and Pant
2009). To date, limited propagation studies have been conducted in
the honeybush speciesC. subternata (Mbangcolo 2008).In order to stan-
dardise the methodology for commercial propagation of C. subternata
cuttings, this study was conducted to determine the effect of rooting
media, plant growth regulators, clone, and season on the rooting of
C. subternata cuttings. The standard protocol used by industry served
as control.
2. Material and methods
2.1. Experimental site and plant material
The experiments were conducted at the nursery of the Agriculture
Research Council (ARC) Infruitec-Nietvoorbij, situated in Stellenbosch,
South Africa. The experiments took place between the end of May
2012 and beginning of March 2013. Cyclopia subternata cutting mate-
rials were sourced from a 2-year-old seed orchard situated at Elsenburg
near Stellenbosch. Cuttings were collected during early morning (8:30
to 9:30 am) and kept moist and cool until transported to the working
area.
2.2. Experimental design
The experiments were organised as a completely randomised block
design in a 3 × 4 × 3 factorial arrangement with growth medium,
clone and plant growth regulator (PGR) as main effects. For each treat-
ment combination (medium × clone × PGR), eight terminal semi-
hardwood cuttings were treated, and this was replicated three times.
Thus, the experiments utilised 72 cuttings per clone and 288 cuttings
per treatment combination. The trials were repeated at six planting
dates: 30 May, 24 July, 18 September and 14 November (in 2012), and
1 February and 13 March (in 2013).
2.3. Preparation of trays and growth media
Polystyrene seedling trays with dimension of 69 cm × 33 cm ×
8.6 cm (length × width × depth) were surface-sterilised in a Spore
Kill™(12% didecyl dimethyl ammonium chloride) solution. Seedling
trays were filled with three growth media: (1) Canadian peat moss,
sterilised river sand and polystyrene balls (3mix) (1:1:1) (v/v/v);
(2) fermented pine bark and sterilised river sand (Bark mix) (1:1) (v/v)
and (3) Canadian peat moss and sterilised river sand (Peat mix)
(2:1) (v/v). The standard growth medium currently used in honeybush
propagation, i.e. 3mix, was used as control medium. Trays were placed
in a mist bed on top of heated sand beds with a temperature of 25 °C,
and only the temperature of the growth media were measured daily, re-
corded with a thermometer (inserted in each growth medium) during
the rooting period. The thermometers were removed each time when
measuring the temperature and inserted again in the growth media
for the next measurement. No other temperature was monitored in
this study. Misting was scheduled to irrigate for 10 s every 15 min
from 8 am to 4 pm delivering ±3.5 mm of water per day and 30 mm
per week.
2.4. Preparation of stem cuttings and application of growth regulators
Terminal cuttings 80 mm long were cut from semi-hardwood stems
with uniform size of 5 mm width. The lower leaves (50% of total leaves)
of each cutting were removed and the base of each cutting was freshly
trimmed by 0.5 mm. The cuttings were immersed in a 0.2% fungicide so-
lution (Dithane® with active ingredient mancozeb 750 g/kg), to protect
cuttings from fungalinfection, prior to dippingin growth regulators. The
basal 1 cm of cuttings was dipped in one of each of the growth regula-
tors Dip & Root™liquid (10 g/L IBA and 5 g/L NAA), Seradix® B2
(3 g/kg IBA) or Seradix® B3 (8 g/kg IBA) before setting in the growth
media. Seradix® B2 served as control as it is used by the honeybush
industry for propagation of cuttings. Cuttings were examined for root
initiation after 63 days in the mist bed.
2.5. Data collection and statistical analysis
Data collected were the percentage of rooted cuttings and surviv-
al, number of roots and root length. Data were analysed using a two-
way analysis of variance (ANOVA) to test for treatment effects using
the general linear model (GLM) procedure of SAS statistical software
version 9.2. The Shapiro–Wilk test was performed to test for normal-
ity (Shapiro and Wilk 1965). Student's test of least significance was
calculated at the 5% level of significance to compare treatment
means.
3. Results
3.1. Rooting medium
3.1.1. Survival and rooting
The rooting medium had a significant effect (p≤0.05) on the
percentage rooting and survival of the cuttings (Table 1). There
were no significant differences between the Bark mix and 3mix. Ap-
proximately 90% of the cuttings planted in these media survived, as
measured 63 days after the start of the treatment. The percentage
rooting of both treatments was in excess of 70%. Cuttings grown
on peat mix showed the lowest survival and rooting, 79.8% and
51.3%, respectively.
3.1.2. Root number and length
The rooting medium had a significant effect (p≤0.05) on the mean
number of roots per cutting and the mean root length (Table 1). Signif-
icantly higher number of roots were recorded on Bark mix (4.7), and
3mix (4.5), compared to Peat mix (2.7). Bark mix also gave the longest
roots (182.8 mm), compared to 3mix (160.9 mm) and Peat mix
(52.6 mm).
Table 1
Effect ofgrowth medium on rootingof C. subternata stemcuttings 63 days after the startof
treatment.
Growth
medium
Survival
percentage (%)
Rooting
percentage (%)
Mean number
of roots
Mean root
length (mm)
Bark mix 90.54a 76.44a 4.86a 182.85a
3mix 89.31a 73.56a 4.84a 160.89b
Peat mix 79.77b 51.27b 2.74b 92.67c
Means with the same letterin the same column are not significantly different (p≤0.05).
76 G.S. Mabizela et al. / South African Journal of Botany 110 (2017) 75–79
3.2. Plant growth regulators (PGRs)
3.2.1. Survival and rooting
The PGRs significantly affected (p≤0.05) the survival rate and
rooting percentage (Table 2), with Seradix® B2 and Seradix® B3 being
the more and similarly effective at ca. 88% survival rate and 69% rooting.
Dip & Root™growth regulator was less effective at 84.1% and 63.9%,
respectively.
3.2.2. Root number and length
The PGRs had a significant effect (p≤0.05) on the number of roots
and length, formed on C. subternata cuttings (Table 2). As for survival
and rooting, Seradix® B2 and Seradix® B3 treatments were similarly ef-
fective in stimulating the formation and growth of roots, while being
more effective than Dip & Root™. The mean number of roots for the
Seradix® treatments was 4.3 mm compared to 3.8 mm for Dip &
Root™. The root length of cuttings treated with Seradix®, irrespective
of the concentration of IBA, exceeded 150 mm, while Dip & Root™
delivered roots of 128.5 mm.
3.3. Clonal effect
3.3.1. Survival and rooting
There were significantdifferences in survival of rooted stem cuttings
(p≤0.05) (Table 3). The highest survival was noted in clones SGD7
(97.5%) and SGD1 (95%). The survival rate of SGD9 (91.2%) was slightly
less, but that of SGD6 (62.3%) substantially lower. The same trend for
the clones was observed in terms of rooting, but in this case SDG7
(83.7%) attained a significantly higher rooting percentage than both
SGD1 (74.7%) and SGD9 (73.4%). Once again, clone SGD6 performed
the poorest with only 36.4% rooting.
3.3.2. Root number and length
Cloning had a significant effect (p≤0.05) on root number and length
(Table 3). The highest root number was recorded for clone SGD9 (5.4).
The numberof roots formed by SGD7 (4.8) and SDG1 (4.4) were not sig-
nificantly different. SGD6 formed substantially less roots (1.9). The lon-
gest roots were formed by SGD7 (198.1 mm) and SGD9 (183.0 mm),
followed by SGD1 (120.8 mm) and SGD6 (79.9 mm) (p≤0.05).
3.4. Seasonal effect
3.4.1. Survival and rooting
Seasonal variation had a significant effect (p≤0.05) on the survival
and rooting percentage of C. subternata cuttings (Table 4). A significantly
higher survival rate was observed for cuttings made during November
(92%) and March (93%) than during May (87%). The survival rates of cut-
tings made during September (83%) and February (84.6%) were slightly,
but significantly, lower than that observed for November and March.
ThelowestsurvivalratewasrecordedduringJuly(77.9%).
The highest rooting percentages (p≤0.05) were recorded for plant-
ing dates from November and March (74.7% and 74.3%, respectively),
followed by May, July and September (66.8%, 66.6% and 63.7%, respec-
tively), and lastly, February (56%) (Table 4). Propagation in May, July
and September would deliver more or less the same results.
3.4.2. Root number and length
The number of roots and length were significantly affected (p≤0.05)
by the seasonal variation (Table 4). A significantly higher root number
was recorded during September (4.5) and March (4.7) compared to
other studied planting dates. The root numbers during November
(4.1), February (3.8) and May (3.6) were not significantly different
from each other. Furthermore, the lowest root number was obtained
for cuttings set during July (3.5), although it was not significantly
different (p≤0.05) from February (3.8).
The longest root length (p≤0.05) was recorded for setting of
cuttings during September (218.7 mm), followed by May (171.3 mm)
and July (159.9 mm). Setting in November (139.8 mm), March
(109.0 mm) and February (74.4) progressively delivered cuttings with
shorter roots.
4. Discussion
4.1. Rooting medium
Rooting medium is one of the most important factors affecting the
rooting of cuttings, and this study provided evidence of the effect of
the composition of growth media on the rooting success of honeybush
(C. subternata) plants. The highest cutting survival and rooting rate,
number of roots and length of roots were achieved on Bark mix and
3mix. Root initiation of C. subternata was considerably lower in the
Peat mix. A number of factors interacting within a growth medium are
known to affect the success of rooting including oxygen, water and nu-
trient availability (Alikhani et al. 2011; Bhardwaj 2014). The success in
improved root initiation of C. subternata using Bark mix and 3mix
could be attributed to the positive interaction of aeration and water-
holding capacity, as compared to Peat mix. A well balanced oxygen
and water-holding capacity promotes oxygen availability, transpiration,
nutrient uptake, growth and aeration during root initiation. Poor aera-
tion is a fundamental problem in waterlogged situations, leading to
decay of cuttings before root initiation (Schmitz et al. 2013). Both Bark
mix and 3mix might had a lower water-holding capacity based on the
composition of the growth media (coarse and semi-coarse texture, re-
spectively) compared to Peat mix (fine-textured and small pores)
(Relf 2009). Creating a good aerated environment will increase respira-
tion at the base of the cuttings, as explained by Akakpo et al. (2014),
Table 2
Effect of growth regulator on rooting of C. subternata stem cuttings 63 days after start of
treatment.
Growth
regulator
Survival
percentage (%)
Rooting
percentage (%)
Mean number
of roots
Mean root
length (mm)
Dip & Root™84.10b 63.93b 3.78b 128.52b
Seradix® B2 87.84a 69.03a 4.35a 151.25a
Seradix® B3 87.67a 68.31a 4.31a 156.64a
Means with the same letter inthe same column are not significantly different (p≤0.05).
Table 3
Clonal effect on rooting of C. subternata stem cuttings 63 days after start of treatment.
Clone Survival
percentage (%)
Rooting
percentage (%)
Mean number
of roots
Mean root length
(mm)
SGD1 95.12a 74.74b 4.38b 120.82b
SGD6 62.26c 36.49c 1.97c 79.89b
SGD7 97.53a 83.71a 4.83b 198.10a
SGD9 91.23b 73.41b 5.40a 183.08a
Means with the same letter inthe same column are not significantly different (p≤0.05).
Table 4
Effect of planting date on rooting of C. subte rnata stem cuttings 63 days after start of
treatment.
Planting date
(months)
Survival
percentage (%)
Rooting
percentage
(%)
Mean number
of roots
Mean root
length (mm)
May 87.45b 66.89b 4.12b 174.32b
July 77.83d 66.66b 3.59c 159.41b
September 83.18c 63.79b 4.51a 218.70a
November 92.12a 74.76a 4.11b 139.88c
February 84.60c 56.13c 3.78bc 74.41e
March 93.98a 74.30a 4.76a 109.11d
Means with the same letterin the same column are not significantly different (p≤0.05).
77G.S. Mabizela et al. / South African Journal of Botany 110 (2017) 75–79
who indicated that rice husk growth medium enhanced rooting of
Vitellaria paradoxa stem cuttings.
Heating of the growth medium mayhave contributed to the rooting
success of stem cuttings. During this study, bottom heating maintained
at 25 °C was used and a thermometer was used to determine the tem-
perature of the growth media (only a single hole on a seedling tray
was monitored). A high rooting percentage was observed at rooting me-
dium temperature of 22 °C–27 °C (Bark mix and 3mix) and low rooting
was observed at growth medium temperature of 17 °C–23 °C (Peat
mix). The differences in the composition of the growth media might ex-
plain differences in temperature between the growth media. A coarse
growth medium retains more heat than a fine-textured growth medium
(Al-Kayiem et al. 2015). Growth medium temperature is known to
be the critical factor in rooting of cuttings. Keeping the rooting medium
extra warm not only increases the speed of rooting but also improves
the percentage of rooted cuttings (Runkle 2006). Reuveni and
Castoriano (1993) treated semi-hardwood cuttings of mango cultivars
with bottom heating of 20 °C, 25 °C, 30 °C and 35 °C and observed higher
rooting at 25 °C and 30 °C, while at 20 °C, cuttings took longer to reach a
high rate of rooting. Furthermore, Runkle (2006) stated that the rate of
root and shoot development is accelerated by growth medium temper-
ature. Given the findings of Runkle (2006) and our observations in
terms of temperature, investigation of the effect of bottom heating on
rooting percentage during plant propagation of C. subternata via stem
cuttings should be investigated in the future.
4.2. Plant growth regulators (PGRs)
There were distinct differences in survival rate, rooting percentage,
number of roots and root length due to the different growth regulators
applied in therooting of C. subternatacuttings. PGRs such as IBA, IAA and
NAA are known to accelerate the rate of rooting and increase final
rooting percentage and number of roots on cuttings (Gehlot et al.
2014; Ibrahim et al. 2015). In the rooting of C. subternata cuttings, the
highest rooting percentage was achieved with Seradix® B2 and
Seradix® B3 (3 and 8 g/kg IBA respectively), compared to Dip &
Root™(10 g/L IBA and 5 g/L NAA). Successful rooting could possibly
be associated with the IBA content in the growth regulators used. In ad-
dition, the number of roots and root length was significantly improved
by the IBA (Seradix® B2 and B3) as well. In plant species such as in
Lippia javanica L. (Soundy et al. 2008)andV. paradoxa C.F. Gaerth
(Akakpo et al. 2014), a high rooting percentage was achieved when
stem cuttings were treated with IBA. Similar results were also obtained
by Carvalho et al. (1995) and Chalapathi et al. (2001) who reported an
increased rooting, number of roots per cutting, root length, root thick-
ness, fresh and dry weight of roots using IBA. The application of Dip &
Root™resulted in lower rooting success, which could possibly be attrib-
uted to the presence of NAA as root growth regulator. In studies where
IAA and NAA were used independently as rooting agent, low percentage
rooting in Bougainvillea glaba cuttings (Singh et al. 2011b)andFicus
hawaii (Hassanein 2013) were reported. Nelson et al. (1992),using
four different auxins (IAA, IBA, NAA and indole-3-propionic acid) in
Pinus taeda,Pinus elliotti var. ellioti and Pinus palustris proved that IAA
and IBA assisted in high root initiation, while rooting with NAA and
indole-3-propionic acid resulted in poor rooting of cuttings of these
species.
4.3. Clonal effect
The effect of clonal variation in rooting response has been reported
by various research studies. The four C. subternata clone selections
from Groendal were selected based on the preliminary results from a
previous study indicating that SGD1 and SGD7 had more than 80%
rooting, followed by SGD6 with 50% to 70% rooting and, SGD9 with
less than 50% rooting (unpublished data). However, the results from
this study indicate that clone SGD7 had more than 80% rooting, SGD1
and SGD9 more than 70% rooting, and SGD6 less than 40% rooting.
Rooting percentage, survival rate, root number and root length were
higher in clones SGD1, SGD7 and SGD9, than in SGD6. The clonal differ-
ences in rooting indicate large differences in rootability and consider-
able genetic variation among clones (Prat et al. 1998). Baltunis and
Brawner (2010) reported that the genetic variation among clones was
statistically significant for rooting of Pinus radiata clonal populations.
4.4. Seasonal effect
A higher survival rate, rooting percentage and number of roots were
attained from cuttings collected in September (spring) and November
(early-summer) and March (autumn) compared to cuttings collected
in July (winter) and February (late summer). This result corroborates
the findings of other studies showing that time of collection as a major
factor affecting vegetative propagation in plant species (Soundy et al.
2008; Haile et al. 2011). Mbangcolo (2008) suggested that the best
time for collecting cuttings of C. genistoides and C. intermedia was during
summer (December). Increased rooting during spring and summer
months may be attributed to higher temperatures and longer illumina-
tion/daylight periods which leads to a higher photosynthetic rate and
carbohydrate reserve content, thereby stimulating adventitious root
development (Rapaka et al. 2005).
The carbohydrate accumulation is also expected to be high in cut-
tings collected during the dry periods (Mediterranean climate) due to
high carbohydratereserves as the result of lower physiological activities
of the donor plants than in May cuttings just after leaf flushing. There-
fore, cuttings collected during February and March are expected to
root easily due to mobilisation of high amounts of carbohydrates and
other metabolites (Haile et al. 2011). However, in this study, cuttings
collected during February had on average lower rooting percentage,
survival rate, number of roots and length of roots. The lower rooting
of C. subternata during February could possibly be due to lower growth
activityor depletion of growthhormones (probably IBA) or other phys-
iological factors after flowering (thin and soft plant material). Seasonal
effect on rooting and shooting of cuttings is high in plants that undergo
a typical low growth activity (lean period)during cold months (winter)
(Leakey 2004).
In the present study, we demonstrated that the standard industry
protocol for rooting of cuttings (3mix and Seradix® B2) yielded good
results and is still recommended as the preferred protocol. Due to the
differences in rooting success of the various C. subternata clones and
the effect of growth media, PGRs and planting date has on rooting
success, it is therefore recommended that rooting of the differentclones
be optimised before commercialisation.
Acknowledgements
The authors acknowledge the National Research Fund (NRF),
Department of Science and Technology (DST), Monetary Treasury
Economic Fund (MTEF), ARC and Tshwane University of Technology
(TUT) for financial support. Special thanks to Dr. Mardé Booyse for her
valuable assistance with the statistical design and analysis, Ms. Marlise
Joubert for assisting with plant material as well as Ms. Veronica Korkie
and Ms. Gaynor Peterson for assisting with the experiments.
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