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South African Journal of Botany 000 (2022) 1 7

Contents lists available at ScienceDirect

South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Seed germination and in vitro propagation of three threatened endemic

South African Aloe species
S.O. Amooa,b,c,*, N.A. Hlatshwayoa,d, Karel Dole
zale,f, J.O. Olowoyod
a
Agricultural Research Council-Vegetables, Industrial and Medicinal Plants, Private Bag X293, Pretoria, South Africa
b
Indigenous Knowledge Systems Centre, Faculty of Natural and Agricultural Sciences, North-West University, Private Bag X2046, Mmabatho 2735, South Africa
c
Department of Botany and Plant Biotechnology, Faculty of Science, University of Johannesburg, P.O. Box 524, Auckland Park 2006, South Africa
d
Department of Biology and Environmental Sciences, School of Science and Technology, Sefako Makgatho Health Sciences University, P. O. Box 60, Medunsa 0204,
South Africa
e

Department of Chemical Biology, Faculty of Science, Palacky University, Slechtitelu

27, Olomouc CZ-783 71, Czech Republic
f

Laboratory of Growth Regulators, Institute of Experimental Botany AS CR, Slechtitelu

27, Olomouc CZ-783 71, Czech Republic

A R T I C L E I N F O A B S T R A C T

Article History: Aloe modesta Reynolds (endangered), A. peglerae Scho € nland (critically endangered), and A. reitzii Reynolds
Received 30 December 2021 (vulnerable) are endemic South African Aloe species valued for their horticultural and/or medicinal value,
Revised 11 April 2022 appearing on the Red List of South African Plants. Propagation is an important step in the cultivation of these
Accepted 19 April 2022
species for their conservation. This study examined the effect of temperature (15, 20, 25, and 30°C) and pho-
Available online xxx
toperiod (constant dark, 16 h light, and constant light) on seed germination of these three species. The in vitro
Edited by O.A. Aremu propagation of A. reitzii was also investigated by examining the effect of different cytokinins [kinetin, 6-ben-
zyladenine (BA), meta-topolin (mT), and meta-topolin riboside (mTR)] on shoot multiplication. Germination
Keywords:
percentage and mean germination rate of A. modesta were significantly inhibited when the seeds were incu-
Aloe
bated at a low temperature (15°C) in comparison to incubation at other temperatures (20 30°C). An
Endangered species
increase in temperature significantly decreased mean germination time (MGT), as well as increased seedling
Micropropagation
Plant tissue culture chlorophyll and carotenoid contents in A. peglerae and A. reitzii. A reduction in germination speed, evident in
Seed propagation increased MGT was observed in the three species when seeds were incubated under constant light and 16 h
Topolins light as compared to constant dark. Medium with 5.0 mM meta-topolin produced the highest A. reitzii shoot
proliferation (16 shoots) per shoot-tip explant after eight weeks of culture. This shoot multiplication rate
translates to a potential production of 1 118 481 shoots per shoot-tip explant per annum, based on a geomet-
ric progression with six possible multiplication cycles per annum. Aloe reitzii shoots produced from BA and
kinetin treatments had a high flavonoid content whereas all mT and mTR treatments gave reduced flavonoid
content. Seed and/or in vitro propagation protocols of these threatened endemic Aloe species established in
this study can be employed in the biodiversity conservation of Aloe species for their sustainable use.
© 2022 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction and horticultural significance (Cousins and Witkowski, 2012;

The Aloe genus (family: Xanthorrhoeaceae) currently consists of
more than 500 accepted species distributed worldwide (WFO, 2021),
the majority of which are found on the African continent
(Amoo et al., 2014). South Africa has the highest number of recog-
nised Aloe species on the African continent (Cousins and Witkow-
ski, 2012) with 153 species currently on the Red List of South African
Plants (SANBI, 2020). The high Aloe species diversity in South Africa
contributes to the country's rich plant diversity, and they are of eth-
nomedicinal, taxonomic, chemical/chemotaxonomic, ecotouristic
* Corresponding author at: Agricultural Research Council-Vegetables, Industrial and
Medicinal Plants, Private Bag X293, Pretoria, South Africa.
E-mail address: amoos@arc.agric.za (S.O. Amoo).
Amoo et al., 2014). In recent times, the collection of these succulents
has attracted a wide range of interest from plant collectors and scien-
tists, eliciting international popularity in the medicinal and horticul-
tural trade industries while exacerbating population decline
(Amoo et al., 2012; Cousins and Witkowski, 2012; Amoo et al., 2014).
Other threats to Aloe species include habitat loss and degradation,
overgrazing, over-zealous or indscirimate collection and over-utilisa-
tion of plant parts that significantly affect the survival of wild species
(Grace, 2011; Cousins and Witkowski, 2012).
Aloe modesta Reynolds, A. peglerae Scho € nland, and A. reitzii Rey-
nolds are among the threatened endemic South African Aloe species
with their conservation status described as vulnerable (endangered
according to Klopper et al. 2020), critically endangered, and vulnera-
ble, respectively, on the Red List of South African Plants (SANBI, 2020).

https://doi.org/10.1016/j.sajb.2022.04.033
0254-6299/© 2022 SAAB. Published by Elsevier B.V. All rights reserved.

Please cite this article as: S.O. Amoo, N.A. Hlatshwayo, K. Dole
zal et al., Seed germination and in vitro propagation of three threatened endemic
South African Aloe species, South African Journal of Botany (2022), https://doi.org/10.1016/j.sajb.2022.04.033
JID: SAJB
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S.O. Amoo, N.A. Hlatshwayo, K. Dolez
al et al.
Aloe modesta is a small, grass-like species found in grassy habitats and
the only endemic Aloe species with sweet-scented flowers (van der
Riet, 1977; Klopper et al., 2020). This species has a specific habitat
requirement with isolated subpopulations in regions with cold, dry
winters and high summer rainfall with moderate temperatures
(van der Riet, 1977; Klopper et al., 2020). Aloe peglerae is a slow-
growing species that is highly exploited and illegally collected for
horticultural trade, resulting in the decimation of its wild populations
at an alarming rate (Cousins and Witkowski, 2012; Pfab et al., 2016).
Reports indicated its ethnomedicinal uses for wound-healing, burns
and infection treatment, and as a laxative (George et al., 2001;
Cock, 2015). Regular fires often scorch the plants in their grassland
natural habitat (Van Wyk and Smith, 2014). Aloe reitzii Reynolds var.
reitzii occurring in montane grassland has a declining population
trend due to habitat degradation from mining activities and afforesta-
tion affecting its restricted geographical range (Mtshali et al., 2018a).
Aloe reitzii Reynolds var. vernalis D.S. Hardy is confined to a single
small area in the KwaZulu-Natal Province, South Africa (Mtshali et al.,
2018b; Klopper et al., 2020). This species is threatened by its wild
harvesting for medicinal purposes and damage caused by baboons
feeding on its flowers (Mtshali et al., 2018b; Klopper et al., 2020).
Propagation and cultivation can reduce the current exploitation
and/or harvesting pressures on wild Aloe populations (Amoo et al.,
2014). Propagation is generally achieved through seeds and/or vege-
tatively for Aloe species. Seed propagation is more feasible and rec-
ommended for survival of rare taxa (Bairu et al., 2009). However,
factors such as the availability of viable seeds, extremely low germi-
nation rates, and reduced long-term survival of seedlings are some of
the limiting factors affecting the propagation of Aloe species
(Bairu et al., 2009; Symes, 2012; Cousins et al., 2013). Environmental
factors including light and temperature can significantly influence
seed germination in Aloe species (Kulkarni et al., 2014). The current
study was therefore designed to investigate the effect of temperature
and photoperiod on the seed germination of three endemic South
African Aloe especies, which are A. modesta, A, peglerae and A. reitzii.
The applicability of adapting previously developed plant tissue cul-
ture protocols for Aloe species (Bairu et al., 2007; Amoo et al., 2012;
Hlatshwayo et al., 2020) was explored for in vitro multiplication of A.
reitzii. The findings from this study will assist in the cultivation of
these species thereby leading to their abundance.
2. Material and methods
2.1. Seed germination
Aloe modesta, A. peglerae, and A. reitzii seeds were purchased from
Silverhill Seeds (Cape Town, South Africa), a recognised seed supplier
dealing with indigenous South African species. The seeds were ger-
minated in clean and unused 9 cm Petri dishes lined with a double
layer of Whatman No. 1 filter paper, moistened initially with 10 ml of
distilled water. Thereafter, small volumes of distilled water were
added when necessary. A seed lot of 25 seeds per Petri dish replicated
four times was used for each treatment. To determine the effect of
temperature on seed germination, the seeds were incubated in
growth chambers at constant temperatures of 15, 20, 25 and 30°C
under 16/8 h light/dark (16 h photoperiod) conditions. For the deter-
mination of the effect of photoperiod on seed germination, the seeds
were incubated under constant 24 h dark, constant 24 h light and
alternating 16:8 h light/dark conditions at a temperature of 25 § 2°C.
Germination trials were considered complete once there were no
seed germination observed for a period of three days or more. Aloe
modesta, A. peglerae, and Aloe reitzii seeds were incubated for a period
of 14, 15, and 20 days, respectively. Germination time (GT) was calcu-
P
lated using the following equation: GT = (n x d)/N, where
n = number of seeds germinated each day, d = number of days from
the beginning of the test and N = total number of seeds germinated at
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South African Journal of Botany 00 (2022) 1 7
the termination of the experiment (Kulkarni et al., 2014). Germina-
tion rate (GR) was calculated based on the following equation: GR (%
P
d 1) = [(G1/t) + (G2/t) + (Gt/t)], where G is the seed germination in
percentage at 1-day intervals and t is the total number of days of the
germination period (Kulkarni et al., 2014). Germination percentage
(GP) was calculated as follows: GP = (Total number of germinated
seeds in the seed lot / Total number of seeds in the seed lot) x 100
(Kader, 2005).
Aloe peglerae and Aloe reitzii seedlings that were germinated
under different temperature regimes were quantified for their chlo-
rophyll and carotenoid contents (Lichtenthaler, 1987). A pestle and
mortar was used to crush one hundred milligrams (0.1 g) of fresh
shoot material in 5 ml acetone. The homogenates were centrifuged
using a bench top Hermle Z513 Centrifuge at 3000 U min 1 for 5 min.
The supernatants were then collected and the absorbance read at
470 nm, 645 nm and 662 nm using a SpecordÒ 210 plus spectropho-
tometer. Each determination was done in triplicate and the statisti-
cally analysed data expressed in microgram per gram (mg/g) fresh
weight. Parameters were computed as follows (Lichtenthaler, 1987):
Chlorophyll a (Ca) = 11.24A662 - 2.04A645;
Chlorophyll b (Cb) = 20.13A645 - 4.19A662;
Total chlorophyll = 7.05A662 + 18.09A645;
Total carotenoids = (1000A470 1.90Ca 63.14Cb)/214.
2.2. Aloe peglerae seedling growth
Aloe peglerae seedlings obtained from seeds incubated at 25°C and
16 h photoperiod during the germination study were transplanted
into small pots to determine the effect of KelpakÒ application on
seedling growth. KelpakÒ is a commercial, liquid seaweed extract
containing auxins, cytokinins, amino acids, and mineral elements,
amongst others (Lo € tze and Hoffman, 2016; Kocira et al., 2020).
Guided by the manufacturer’s recommendations, the pots were soil
drenched with six dilutions (5, 15, 25, 35, 45 and 55 ml/L) of KelpakÒ
solution, a commercial liquid seaweed concentrate. There were 25
seedlings per treatment. A control group with no KelpakÒ added was
incorporated into the experiment. KelpakÒ treatments were added to
the pots once every four weeks for 12 weeks. The experiment was
conducted using a completely randomised block design in a glass-
house with natural lighting and an average temperature of 25 § 2°C.
At the end of the experiment, the shoot length, number of leaves,
shoot fresh weight, length of longest root, and number of roots were
recorded.
2.3. In vitro propagation of Aloe reitzii
Aloe reitzii seeds were surface-decontaminated and germinated in
vitro on one-tenth strength Murashige and Skoog (MS) medium as
previously described (Hlatshwayo et al., 2020). Therafter, bulking of
explants was carried out on MS medium (Murashige and Skoog, 1962)
containing 0.1 mg/L thiamine HCl, 0.5 mg/L pyridoxine HCl, 0.5 mg/L
nicotinic acid, 2.0 mg/L glycine, 100 mg/L myo-inositol, and 3% (w/v)
sucrose. After adjusting the pH to 5.7, the medium was solidified
with 0.8% (w/v) agar (Bacteriological agar - Oxoid Ltd., Basingstoke,
Hampshire, England) and autoclaved at 121°C and 103 kPa for
20 min. The cultures were incubated in a growth room set at 25 § 2°
C and alternating 16:8 h light/dark conditions. The effect of cytoki-
nins on shoot multiplication was evaluated using kinetin, 6-benzyla-
denine (BA), meta-topolin (mT), and meta-topolin riboside (mTR),
each at 2.5, 5.0, 7.5, 10.0 and 15.0 mM concentrations. A plant growth
regulator-free medium served as the control. A combination of 5.0
mM meta-topolin (being the optimum cytokinin concentration giving
the highest number of shoot per explant from the cytokinin experi-
ment) was combined with different concentrations (0.0, 1.0, 2.0, and
5.0 mM) of indole-butyric acid (IBA) in another experiment in order
to determine the effect of cytokinin and auxin interaction on shoot
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proliferation. In these experiments, each shoot-tip explant (0.5 to
1.0 cm in length; Fig. 1) was cultured on 40 mL medium in screw-cap
jars (110 mm x 60 mm, 300 ml volume). Each experiment was con-
ducted twice with 20 replicates per treatment in a completely ran-
domized design. After eight weeks of culture in each experiment, the
total number of shoots per explant, number of shoots less than
1.0 cm in length, number of shoots greater than or equal to 1.0 cm in
length, frequency of rooting, and shoot fresh weight were recorded.
Relatively big in vitro proliferated shoots (with shoot length
greater than 1.0 cm) were cultured on half-strength MS medium
with 0.0, 1.0, 2.5 or 5.0 mM of IBA or naphthalene acetic acid (NAA)
for in vitro rooting under the same culture conditions used for the
preceding experiments. After six weeks of culture, the number of
roots per cultured shoot, rooting frequency, length of the longest root
and number of additional shoots produced per cultured shoots were
recorded. Agar was carefully washed off the rooted shoots following
their removal from the rooting medium, and the shoots were potted
in a 1:1 (v/v) mixture of sand and soil, kept moist with daily light
watering. The plantlets were acclimatised in a glasshouse with natu-
ral lighting and an average temperature of 25 § 2°C for a period of 6
weeks. On a weekly basis, the survival frequencies of acclimatized
plantlets were recorded over a six-week period.
The total phenolic and flavonoid contents were quantified at the
end of the in vitro shoot proliferation experiment. The randomly sam-
pled plant materials (oven-dried at 50°C) from each treatment were
ground to fine powder and extracted following the method outlined
by Makkar (2000). Extraction of the ground plant materials (0.2 g)
was done using 50% aqueous methanol (10 ml) in a sonication bath
for 20 min. The slightly modified Folin and Ciocalteu method outlined
by Fawole et al. (2009) was used to quantify the total phenolic con-
tent with gallic acid used to plot the calibration curve. Results were
expressed in mg gallic acid equivalents (GAE) per g dry weight (DW).
For flavonoid content quantification, the aluminium chloride colori-
metric method was used (Zhishen et al., 1999) with the calibration
curve plotted using catechin. Flavonoid content was expressed in mg
catechin equivalents (CE) per g DW. Each determination was ana-
lysed in triplicate.
2.4. Data analysis
The data collected were subjected to one-way analysis of variance
(ANOVA) using SPSS statistical software (version 16.0). At p = 0.05,
significant differences were established followed by a posthoc mean
value separation using Duncan’s Multiple Range Test.
Fig. 1. Shoot-tip explant (A), in vitro rooting of proliferated shoot indicating additional shoot production and healthy root development (B), and fully acclimatized Aloe reitzii plants.
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South African Journal of Botany 00 (2022) 1 7
3. Results and discussion
3.1. Seed germination
Temperature significantly influenced the mean germination per-
centage (MGP), mean germination time (MGT) and mean germina-
tion rate (MGR) of A. modesta seeds (Table 1). Both MGP and MGR
were significantly inhibited when the seeds were incubated at a low
temperature (15°C) compared to incubation at other temperatures
(20 30°C). In the case of A. peglerae and A. reitzii, temperature signifi-
cantly influenced their MGT, while the MGP and MGR were not sig-
nificantly affected (Table 1). An increase in temperature significantly
resulted in a decrease in MGT. The lower the MGT, the faster the ger-
mination. The species exhibited varied response as A. modesta
showed no significant difference in MGT at high temperatures (20
30°C), while A. peglerae and A. reitzii showed significant difference in
MGT at 25 or 30°C. Thus, when considering all the three germination
parameters, seed incubation at 25°C more favourably promoted seed
germination of A. peglerae and A. modesta. Seed germination at 25°C
is considered to be a suitable or optimum temperature for many spe-
cies including desert and semi-desert shrubs; hence many commer-
cial species are germinated at this temperature (Baskin and
Baskin, 1998). Moderate temperatures (25 30°C) similarly
improved seed germination of some Aloe species, including A. bosca-
wenii, A. ferox, and A. lateritia (Abihudi et al., 2020; Bairu et al., 2009).
Temperature remains an extremely important climatic and environ-
mental factor that affects seed germination in many plant species
(Bairu et al., 2009; Kulkarni et al., 2011) and significantly influences
the geographical distribution of Aloe species (Cousins and Witkow-
ski, 2012).
Aloe peglerae seedlings that were raised from seeds incubated at
25°C contained significantly higher levels of chlorophyll a, total chlor-
ophylls and total carotenoids when compared to seedlings raised at
lower temperatures (Table 2). Seed incubation at 30°C was more
favourabe for A. reitzii seed germination as it resulted in significantly
increased germination speed (Table 1), as well as an increase
(although non-significant in some cases) in chlorophyll a, total chlor-
ophylls and total carotenoids contents when compared to other tem-
peratures (Table 2). Chlorophylls are important pigments in
chloroplasts that play an important role in light harvesting for the
photochemical process in photosynthesis (Richardson et al., 2002).
Thus, low chlorophyll contents resulting in reduced photosynthetic
potential can limit plant primary production (Richardson et al.,
2002). On the other hand, temperature can significantly influence
plant pigments including chlorophyll and carotenoid contents in
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Table 1
Effect of temperature on the mean germination time, mean germination rate and mean germination percentage of Aloe peglerae (AP), Aloe reitzii (AR) and Aloe
modesta (AM).
1
Temperature (°C) Mean germination time (day) Mean germination rate (% day ) Mean germination percentage (%)

AP AR AM AP AR AM AP AR AM

15 10.0 § 0.1 a 11.1 § 0.2 a 4.0 § 0.3 b 6.3 § 0.1 a 1.8 § 0.1 a 2.4 § 0.3 b 95.0 § 1.9 a 35.0 § 2.5 a 33.0 § 3.5 b
20 7.1 § 0.1 b 8.3 § 0.1 b 9.1 § 0.4 a 6.3 § 0.2 a 2.0 § 0.4 a 6.2 § 0.1 a 95.0 § 3.0 a 40.0 § 7.8 a 87.0 § 1.3 a
25 6.6 § 0.2 c 7.0 § 0.2 c 8.2 § 0.4 a 6.5 § 0.1 a 1.7 § 0.2 a 6.2 § 0.3 a 98.0 § 1.2 a 34.0 § 4.8 a 91.0 § 4.8 a
30 7.5 § 0.4 b 3.1 § 0.1 d 8.2 § 0.3 a 6.4 § 0.1 a 2.0 § 0.1 a 6.3 § 0.2 a 96.3 § 1.0 a 39.0 § 1.0 a 88.0 § 2.3 a
Mean values within the same column with different letters indicate significant difference (P = 0.05) based on Duncan’s Multiple Range Test. n = 4.

Table 2
Effect of temperature on chlorophyll and carotenoid contents of Aloe peglerae and Aloe reitzii seedlings.

Plant species Temperature Chlorophyll and carotenoid contents (mg/g FW)

Chlorophyll a Chlorophyll b Total chlorophylls Total carotenoids Chlorophyll(a+b) / Total carotenoids

Aloe peglerae 15°C 103.1 § 15.4 c 119.2 § 27.6 ab 222.3 § 43.0 b 34.9 § 2.8 c 6.3 § 0.7 a
20°C 156.6 § 8.6 b 93.1 § 10.9 b 249.7 § 19.6 b 44.3 § 2.4 b 5.6 § 0.2 a
25°C 227.3 § 9.8 a 160.5 § 2.8 a 387.8 § 9.4 a 61.1 § 0.9 a 6.4 § 0.1 a
30°C 237.6 § 5.6 a 113.0 § 3.9 ab 351.0 § 9.5 a 59.0 § 1.2 a 5.9 § 0.0 a
Aloe reitzii 15°C 114.1 § 20.8 b 91.9 § 2.9 c 206.0 § 22.4 b 33.3 § 2.2 d 6.2 § 0.3 a
20°C 171.2 § 5.8 ab 123.7 § 11.7 b 294.9 § 16.9 a 44.2 § 0.6 c 6.7 § 0.3 a
25°C 157.4 § 27.3 b 184.3 § 10.8 a 341.7 § 25.0 a 49.5 § 0.8 b 6.9 § 0.4 a
30°C 224.6 § 2.2 a 129.9 § 3.6 b 354.3 § 5.8 a 56.3 § 0.1 a 6.3 § 0.1 a
Mean values within the same column with different letters for each species indicate significant difference (P = 0.05) based on Duncan’s Multiple Range Test.
FW = Fresh weight. n = 3.

plants (Taylor and Rowley, 1971). Low temperatures resulted in sig-
nificantly lower chlorophyll and carotenoid contents in A. peglerae
and A. reitzii seedlings (Table 2). Low temperatures (below 20°C) sim-
ilarly resulted in reduced chlorophyll and/or carotenoid contents in
rice, maize and redroot pigweed (Amaranthus retroflexus) seedlings
arguably due to the decline in the synthesis of chlorophyll precursors
or photodestruction of chlorophyll before it becomes stabilized
through complexing with chloroplast lamellae (Guo and Al-Kha-
tib, 2003; McWilliam and Naylor, 1967; Miedema, 1982; Taylor and
Rowley, 1971; Zhao et al., 2020). The accompanied low carotenoid
content at low temperatures could be due to the protective role of
carotenoids against chlorophyll photodestruction (McWilliam and
Naylor, 1967; Miedema, 1982).
Photoperiod, another important environmental factor influencing
seed germination (Bairu et al., 2009), did not significantly influence
MGP and MGR of the three Aloe species (Table 3). Subjecting the
seeds to 24 h dark conditions however, led to significantly lower
MGT for A. peglerae and A. reitzii seeds. A reduction in germination
speed, evident in an increased MGT was observed in all the species
with an increase in light conditions (Table 3). Aloe species present
variations in their need for light to achieve successful germination
(Kulkarni et al., 2014). Unlike the positively photoblastic A. arbores-
cens seeds (Kulkarni et al., 2014), the presence of light delayed germi-
nation speed recorded for all the three Aloe species in the current
study.
3.2. Aloe peglerae seedling growth
Commercial seaweed extracts such as KelpakÒ treatment are
known to improve seedling growth and vigour (Papenfus et al., 2013;
Van Staden et al., 1995). In the current study, KelpakÒ treatment at
45 ml/L promoted shoot length and shoot fresh weight of A. peglerae
without any negative effect on the number of leaves and root produc-
tion (Table 4). Thus, commercial biostimulant extract can be applied
to enhance seedling establishment and initial growth of A. peglerae
under cultivation.
3.3. In vitro propagation of Aloe reitzii
Cytokinin application significantly influenced A. reitzii shoot pro-
liferation (Table 5). An increase in shoot proliferation was observed
with an increase in BA, kinetin, and mT concentrations until the opti-
mal concentration (5.0 mM), beyond which there was a decline in
shoot production with an increase in concentration. This same trend
was observed with mTR treatments but with optimum concentration
at 7.5 mM. The dose-dependent response of shoot-tip explant to cyto-
kinins was also reported in A. arborescens and A. polyphylla tissue cul-
ture protocols (Amoo et al., 2012; Bairu et al., 2007). The highest
shoot proliferation (16 shoots per explant), which was recorded in
the medium with 5.0 mM mT, was approximately six-fold of what
was recorded for the control, albeit the majority of the shoots were

Table 3
Effect of photoperiod on the mean germination time, mean germination rate, and mean germination percentage of Aloe peglerae (AP), Aloe reitzii (AR) and
Aloe modesta (AM).
1
Photoperiod Mean germination time (day) Mean germination rate (% day ) Mean germination percentage (%)

AP AR AM AP AR AM AP AR AM

Constant dark 4.8 § 0.1 b 5.3 § 0.1 c 7.1 § 1.9 a 6.6 § 0.1 a 2.1 § 0.2 a 6.1 § 0.3 a 99.0 § 1.0 a 42.0 § 3.5 a 92.0 § 4.6 a
16 h light 6.3 § 0.1 a 6.7 § 0.0 b 8.2 § 0.4 a 6.4 § 0.1 a 2.5 § 0.2 a 6.0 § 0.3 a 96.0 § 1.6 a 50.0 § 4.2 a 91.0 § 4.8 a
Constant light 6.7 § 0.2 a 8.3 § 0.4 a 9.1 § 0.2 a 6.5 § 0.1 a 2.3 § 0.3 a 6.2 § 0.2 a 97.0 § 1.0 a 46.0 § 6.2 a 93.0 § 3.5 a
Mean values within the same column with different letters indicate significant difference (P = 0.05) based on Duncan’s Multiple Range Test. n = 4.

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Table 4
Seedling growth of Aloe peglerae as influenced by different dilutions of KelpakÒ seaweed concentrate.

KelpakÒ dilution (ml/L) Shoot length (cm) Number of leaves Shoot fresh weight (g) Length of longest root (cm) Number of roots

0 3.1 § 0.2 b 4.6 § 0.3 a 4.0 § 0.6 ab 14.7 § 1.1 a 7.7 § 0.5 a
5 3.0 § 0.1 b 4.6 § 0.2 a 3.5 § 0.4 b 13.7 § 1.1 a 7.6 § 0.4 a
15 3.3 § 0.2 ab 5.3 § 0.2 a 4.8 § 0.6 ab 15.4 § 0.8 a 8.5 § 0.4 a
25 3.2 § 0.2 b 4.7 § 0.3 a 3.9 § 0.6 b 15.0 § 1.0 a 8.1 § 0.5 a
35 3.4 § 0.2 ab 5.2 § 0.3 a 4.8 § 0.6 ab 16.4 § 1.3 a 8.1 § 0.5 a
45 3.7 § 0.1 a 5.2 § 0.2 a 5.7 § 0.5 a 16.7 § 0.8 a 8.7 § 0.4 a
55 3.5 § 0.1 ab 5.2 § 0.2 a 5.0 § 0.6 ab 16.4 § 1.0 a 8.5 § 0.6 a
Mean values within the same column with different letters indicate significant difference (P = 0.05) based on Duncan’s Multiple Range Test. n = 25.

small in size. The size of the shoots produced seemed to be a trade-
off, considering the high number of shoots produced per explant.
However, these small shoots are able to grow to a transplantable size
during the in vitro rooting phase of individual shoot. Although kinetin
(2.5 and 5.0 mM) and mTR (7.5 mM) treatments produced signifi-
cantly high number of shoots greater than 1 cm in length, these treat-
ments produced a significantly less number of shoots per explant in
comparison to mT (5.0 mM). The superiority of mT and/or its deriva-
tives has been established in the micropropagation of other endemic
Aloe species including A. arborescens, A. ferox, A. peglerae and A. poly-
phylla (Amoo et al., 2012; Bairu et al., 2007,2009; Hlatshwayo et al.,
2020). It is noteworthy that the optimum concentration for shoot
proliferation (5.0 mM mT) in the current study was also the optimum
concentration in the plant multiplication protocols developed for A.
arborescens and A. polyphylla (Amoo et al., 2012; Bairu et al., 2007).
Aloe reitzii is harvested for medicinal purposes (Klopper et al.,
2020), underpinned by its constituent secondary metabolites. In vitro
secondary metabolite production offers an alternative approach for
the production of bioactive secondary metabolites and species con-
servation. In vitro plant secondary metabolite production may be
affected by the type of plant growth regulators (Amoo and Van Sta-
den, 2013; Coste et al., 2011; Do €rnenburg and Knorr, 1995;
Hlatshwayo et al., 2020). Shoots produced from treatments with BA
and kinetin had a high flavonoid content whereas all mT and mTR
treatments gave reduced flavonoid content (Table 5). Similarly, mT
treatments resulted in a comparatively reduced total phenolic con-
tent. Nonetheless, the total phenolic content quantified in the
different treatments was higher than the total phenolic content
reported in 6-week old A. arborescens cultures, whereas the flavonoid
content in the current study was lower than what was recorded in
the 6-week old cultures (Amoo et al., 2012).
Combinations of auxins with cytokinins could improve in vitro
shoot proliferation (Amoo and Van Staden, 2013; Coenen and
Lomax, 1997; Rasool et al., 2013). In this study, combinations of opti-
mal cytokinin concentration (5.0 mM mT) with concentrations (1.0,
2.0 or 5.0 mM) of IBA did not significantly improve shoot multiplica-
tion or total phenolic production of the proliferated shoots. Similar
results regarding the lack of significant positive effect of auxin sup-
plementation on shoot production have been reported in A. peglerae
and A. polyphylla (Chukwujekwu et al., 2002; Hlatshwayo et al.,
2020). The combinations with 1.0 mM (yielding 2.88 mg CE/g DW) or
5.0 mM IBA (2.98 mg CE/g DW) resulted in increased flavonoid con-
centrations in the proliferated shoots when compared to the control
(without IBA with 2.34 mg CE/g DW).
Rooting and acclimatization are important stages in establishing
successful micropropagation protocols. Functional rooting of individ-
ual shoots contributes to better acclimatization of in vitro produced
shoots. The use of auxins (IBA and NAA) did not significantly promote
rooting of the in vitro propagated shoots (Table 6). Indeed, all NAA
concentrations inhibited rooting. NAA similarly inhibited in vitro
rooting of A. peglerae (Hlatshwayo et al., 2020). Overall, the control
(without any auxin) had the highest rooting frequency, promoted
rooting, and resulted in significantly higher number of additional
shoot production during rooting (Table 6, Fig. 1). This is particularly

Table 5
Effect of cytokinin types and concentrations on adventitious shoot production and in vitro secondary metabolite production of Aloe reitzii after 8 weeks of culture.

Cytokinin Concentration No. of shoots per No. of shoots Individual regenerated Rooting Total phenolic Flavonoid
(mM) explant (n) <1 cm in length 1 cm in length shoot fresh weight (mg) `frequency concentration `concentration
(mg GAE/g DW) (mg CE/g DW)

Control 0.0 2.78 § 0.49 ij 0.61 § 0.23 i 2.17 § 0.52 cdef 809.98 § 123.32 b 88.89% 11.70 § 0.51 bcde 1.51 § 0.03 efg
BA 2.5 7.72 § 1.24 defg 5.50 § 1.07 def 2.22 § 0.49 bcdef 703.36 § 180.97 b 0% 12.68 § 0.23 bc 2.07 § 0.02 b
5.0 13.39 § 1.54 ab 11.44 § 1.46 b 1.94 § 0.33 def 370.84 § 92.09 b 5.56% 12.21 § 1.56 bcd 2.33 § 0.05 ab
7.5 6.06 § 0.76 fghi 4.35 § 0.65 efg 1.70 § 0.25 ef 1097.70 § 163.25 b 0% 11.34 § 1.61 bcdef 1.94 § 0.02 bcd
10.0 4.67 § 0.72 ghij 3.28 § .62 efghi 1.39 § 0.20 f 1321.80 § 176.43 b 0% 12.98 § 0.73 b 1.63 § 0.06 cde
15.0 2.17 § 0.31 i 0.94 § 0.25 hi 1.22 § 0.10 f 2887.50 § 646.32 ab 0% 11.07 § 0.81 bcdef 2.31 § 0.14 ab
Kinetin 2.5 6.82 § 0.96 efgh 3.29 § 0.53 efghi 3.53 § 0.58 abc 1030.40 § 327.81 b 42.11% 16.04 § 3.40 a 2.36 § 0.04 ab
5.0 10.47 § 1.15 bcde 5.74 § 0.90 def 4.74 § 0.65 a 666.54 § 133.03 b 52.63% 11.98 § 1.02 bcd 2.57 § 0.23 a
7.5 8.20 § 0.73 cdefg 5.70 § 0.60 def 2.50 § 0.35 bcdef 515.00 § 130.09 b 10.53% 13.05 § 1.43 b 2.31 § 0.07 ab
10.0 6.17 § 0.70 fghi 2.67 § 0.54 fghi 3.50 § 0.57 abc 848.34 § 192.97 b 38.89% 13.26 § 2.07 b 2.01 § 0.19 bc
15.0 7.68 § 1.08 defg 5.74 § 0.95 def 1.95 § 0.33 def 666.14 § 240.70 b 0% 11.69 § 0.60 bcde 2.08 § 0.05 b
mT 2.5 13.47 § 1.47 ab 10.53 § 1.17 bc 2.95 § 0.53 bcde 369.96 § 83.03 b 5.88% 10.59 § 0.38 cdef 1.34 § 0.12 efgh
5.0 16.16 § 1.81 a 14.21 § 1.63 a 1.95 § 0.39 def 405.24 § 86.44 b 0% 10.30 § 0.43 def 1.09 § 0.04 gh
7.5 9.53 § 0.95 cdef 7.84 § 0.96 cd 1.68 § 0.28 ef 432.55 § 71.64 b 0% 9.55 § 0.22 ef 1.21 § 0.15 efgh
10.0 5.88 § 1.34 fghi 3.88 § 1.03 efgh 2.00 § 0.48 def 1192.70 § 250.68 b 0% 10.30 § 0.46 def 1.53 § 0.33 ef
15.0 3.45 § 0.49 hij 2.00 § 0.37 ghi 1.45 § 0.18 ef 972.81 § 178.26 b 0% 9.38 § 0.15 f 1.52 § 0.22 ef
mTR 2.5 11.00 § 2.08 bcd 7.63 § 1.57 cd 3.37 § 0.78 abcd 565.81 § 136.51 b 0% 13.29 § 0.33 b 1.13 § 0.06 fgh
5.0 11.21 § 1.34 bcd 8.47 § 1.20 cd 2.74 § 0.49 bcdef 465.20 § 120.41 b 5.26% 11.97 § 0.51 bcd 1.57 § 0.05 de
7.5 11.68 § 1.03 bc 8.00 § 0.83 cd 3.68 § 0.63 ab 5626.60 § 5150.90 a 15.79% 11.96 § 0.61 bcd 1.31 § 0.14 efgh
10.0 8.22 § 1.25 cdefg 5.83 § 0.87 de 2.39 § 0.49 bcdef 621.37 § 169.54 b 0% 10.44 § 0.04 cdef 0.96 § 0.05 h
15.0 5.00 § 0.64 ghij 3.59 § 0.569 efghi 1.41 § 0.19 ef 801.64 § 126.69 b 10% 12.23 § 0.64 bcd 1.40 § 0.03 efg
Mean values within the same column with different letters indicate significant difference (P = 0.05). BA = 6-benzyladenine, mT = meta-Topolin, mTR = meta-topolin
riboside, DW = Dry weight, n = 20 for the proliferation data, n = 3 for determination of total phenolic and flavonoid concentrations.

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S.O. Amoo, N.A. Hlatshwayo, K. Dolez
al et al. South African Journal of Botany 00 (2022) 1 7

Table 6
Effect of auxin types and concentrations on in vitro rooting of proliferated Aloe reitzii shoots after 6 weeks of culture.

Auxin Concentration No. of roots per shoot Length of longest root (cm) No. of additional shoots produced per cultured shoot Rooting frequencies (%)

Control 0.0 mM 5.45 § 0.79 a 5.76 § 0.68 a 5.10 § 1.10 a 100

IBA 1.0 mM 5.20 § 1.04 a 5.68 § 1.00 a 3.50 § 0.72 b 75
2.5 mM 4.45 § 1.05 a 3.66 § 0.66 b 2.15 § 0.67 bc 70
5.0 mM 3.95 § 0.96 a 2.54 § 0.58 b 1.20 § 0.42 cd 55
NAA 1.0 mM 0 0 0.41 § 0.19 cd 0
2.5 mM 0 0 0.84 § 0.49 cd 0
5.0 mM 0 0 0.11 § 0.07 d 0
Mean values within the same column with different letters indicate significant difference (P = 0.05). IBA = Indole-3-butyric acid, NAA = Naphthalene acetic acid, n = 20.

Table 7
Survival frequency of Aloe reitzii plantlets (n = 20) rooted with different auxin concentrations and accli-
matized in a glasshouse.

Auxin type Concentration Survival frequency (%)

Week 1 Week 2 Week 3 Week 4 Week 5 Week 6

Control 0.0 mM 100 80 75 60 55 50

IBA 1.0 mM 100 80 73 66 60 60
2.5 mM 100 93 93 71 43 43
5.0 mM 100 64 9 9 0 0
IBA = Indole-3-butyric acid.

of economic value as it helps to save on the cost of production. Addi-
tional shoots produced during the in vitro rooting phase increase
shoot proliferation of this protocol. Half-strength medium without
any plant growth regulator similarly gave the best rooting response
in A. peglerae and A. polyphylla (Chukwujekwu et al., 2002;
Hlatshwayo et al., 2020). Rooted plantlets from the control medium
were successfully acclimatized with 50% survival frequency recorded
after 6 weeks (Table 7). Nonetheless, the survival frequency is rela-
tively low, and may require further optimization.
4. Conclusions
This study demonstrated the effect of temperature and photope-
riod on seed germination of the three endemic Aloe species. Low tem-
peratures (below 25°C) increased mean germination time and
produced seedlings with reduced chlorophyll and carotenoid con-
tents in A. peglerae and A. reitzii. Incubation at 25°C favoured
enhanced seed germination of A. peglerae and A. modesta while incu-
bation at 30°C favoured A. reitzii. Seeds germinated under constant
dark condition had lower mean germination time, an indication of
faster germination. Previous Aloe species tissue culture protocol was
successfully adapted for the development of A. reitzii micropropaga-
tion protocol. Medium with 5.0 mM mT gave the highest shoot num-
ber (16 shoots) per shoot-tip explant after eight weeks of culture.
This translates to potential production of 1 118 481 shoots per shoot-
tip explant per annum, based on a geometric progression with six
possible multiplication cycles per annum. The acclimatization of A.
reitzii requires optimization in order to improve the survival fre-
quency. Seed and/or in vitro propagation of these threatened endemic
Aloe species established in this study can be employed in the biodi-
versity conservation of these species for their sustainable use.
Declaration of Competing Interest
The authors declared no conflict of interests.
Acknowledgements
Authors gratefully acknowledged institutional supports and fund-
ing from the Agricultural Research Council (ARC), South Africa, Sefako
Makgatho Health Sciences University, National Research Foundation
(Grant UID: 119286), and the Ministry of Education, Youth and Sports
of the Czech Republic, ERDF project "Centre for Experimental Plant
Biology" (No. CZ.02.1.01/0.0/0.0/16_019/0000738).
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