Welehaweria and Sbhatu BMC Research Notes (2023) 16:215 BMC Research Notes

https://doi.org/10.1186/s13104-023-06490-0

R E S E A R C H N OT E Open Access

In vitro micropropagation of Aloe elegans Tod.

using offshoot cuttings
Mebrahtom Welehaweria1 and Desta Berhe Sbhatu2*

Abstract
Objective Aloe elegans Tod. is an ecologically, environmentally, medicinally, and commercially useful aloe species
in Ethiopia and Eritrea. Unfortunately, it is highly threatened due to industrial and urban expansion and traditional
mining and agricultural activities. As a consequence, it is included in the IUCN List of Threatened Species since 2013.
The plant is getting thinly populated in many parts of the Tigrai floristic region since it is being exploited for traditional
and commercial purposes. Therefore, this study was aimed to develop a reproducible, large-scale micropropagation
protocol using offshoot cuttings in Murashige and Skoog (MS) media enriched with plant growth regulators (PGRs).
Results Sterilized explants cultured in full-strength MS media enriched with 0.25 mg/L benzyl amino purine (BAP)
and 0.10 mg/L naphthaleneacetic acid (NAA) resulted in 100% healthy and live (i.e., initiated) explants after four weeks
of initiation study. Unsupplemented initiation media (control) yielded only 14.3% initiated explants. The initiated
explants were tested for their shooting response to produce microshoots by incubating in different concentrations
and combinations of BAP and NAA for four weeks. Fewer days to shooting (13.0 ± 1.0 days), higher mean shoot
number (5.0 ± 1.0), and higher mean shoot length (9.20 ± 2.35 cm) were observed with 1.0/0.50, 1.0/0.25, and 1.0
/0.50 mg/L BAP/NAA combinations, respectively. The rooting responses of the microshoots toward producing
plantlets were also tested by incubating them in half-strength MS media enriched with different concentrations of
NAA and indole-3-butyric acid (IBA) for four weeks. Fewer mean days to rooting (12.0 ± 1.0 days), higher mean root
number (8.0 ± 4.0), and higher mean root length (7.53 ± 3.03 cm) were observed in MS media enriched with 0.75, 0.75,
and 1.25 mg/L IBA, respectively. The responses of A. elegans plantlets to primary (in greenhouse) and secondary (in
nursery shade and direct sunlight) acclimatization in coco peat, composted soil, and manured soil media were high –
with survival percentages of 87.5–97.8% in three to four weeks.
Keywords Acclimatization, Aloe elegans, Initiation, Micropropagation, Rooting, Shooting

*Correspondence:
Desta Berhe Sbhatu
[email protected]
1
Department of Biology, College of Natural and Computational Sciences,
Mekelle University, PO Box 231, Mekelle, Ethiopia
2
Department of Biological and Chemical Engineering, Mekelle Institute of
Technology, Mekelle University, PO Box 1632, Mekelle, Ethiopia

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Welehaweria and Sbhatu BMC Research Notes (2023) 16:215 Page 2 of 7

Introduction DB003/19). Vigorous and healthy looking mother plants

Aloe L. (Aloaceae) comprises the most familiar and use- were located. Then, offshoots were carefully dug out from
ful succulent flowering plants. Ethiopia and Eritrea are the base of the mother plants without causing mechanical
home to 50 species of Aloe of which 31 being endemic. damages and contaminations. Specimens were trimmed
They grow in various altitudes stretching from sea level at to 1.5–2.0 cm long explants for sterilization. Primary and
the Red Sea coast of Massawa, Eritrea (A. eumassawana secondary sterilizations of the explants were carried out
Carter, Gilbert & Sebsebe) to about 3,500 m at Ankober according to standard procedures by using: tap, distilled
of Showa, Ethiopia (A. ankoberensis Gilbert & Sebsebe) and sterilized distilled water; Tween-20 and soap solu-
exhibiting high degree of endemism [1, 2]. A. elegans – a tion; aqueous solution (of 0.25% rocide, 0.25% ridomile
wild species not known anywhere else – grows in rocky and 0.25% bayleton); 5% v/v of NaOCl; and HgCl2 (0.1%
slopes, mostly on sandstone (limestone), in evergreen w/v aqueous solution) [7–9].
bushlands, and wooded grasslands between 1,500 and
2,400 m in Tigrai, Wollo, Gojjam, and Showa floristic Micropropagation experiments
regions in Ethiopia and in Eritrea [1]. The standard procedure of Murashige and Skoog (MS)
Despite their ecological, environmental, traditional (in [10] was employed to prepare sterile growth media
traditional medicine), and commercial benefits, aloes enriched with three plant growth regulators (PGRs),
are highly threatened due to industrial and urban expan- namely benzyl amino purine (BAP), naphthaleneace-
sion and traditional mining and agricultural activities tic acid (NAA), and indole-3-butyric acid (IBA). Initia-
and developments. They are highly endemic and natu- tion and shooting experiments were carried out by using
rally restricted to small geographical areas. Moreover, full-strength MS media while rooting experiment was
they propagate vegetatively very slowly in their natural conducted using half-strength media. Firstly, shoot initia-
environments [1]. Therefore, nearly all species of Aloe, tion experiment was conducted by inoculating 105 steril-
including the Ethiopian aloes, are listed in the CITES ized explants distributed in three groups in full-strength
(Convention on the International Trade in Endangered MS media enriched with 0.25 mg/L BAP and 0.10 mg/L
Species of Wild Fauna and Flora) Appendix II to prohibit NAA in 300 mL magenta culture bottles. Thirty-five (35)
the commercial exploitation of the plants from their wild additional explants were inoculated into full-strength
stands [3, 4]. MS media without BAP and NAA. Each bottle has one
Preliminary studies on its ethnobotany and phyto- explant. Explants were incubated in growth room for
chemistry of its gel have showed that A. elegans has high four weeks for initiation. Secondly, shooting experiment
potential as sources of cosmetic and toiletry, medicinal, was carried out using four shooting media enriched with
and pharmaceutical products [3–5]. However, we have 1.0 + 0.25, 1.0 + 0.50, 1.50 + 0.25 and 2.0 + 0.25 mg/L BAP
observed the plant in many parts of the Tigrai floristic and NAA, respectively, and one non-enriched media
region as thinly populated. For this reason, it is included (control) in four replicates. Initiated explants were har-
in the IUCN List of Threatened Species since 2013 [6]. vested from the initiation media and one initiated explant
Commercial exploitation of the plant will, therefore, was cultured in each culture bottle. The cultured explants
require large-scale cultivation. In line with this rationale, were incubated for four weeks to produce viable micro-
a reproducible protocol for its in vitro propagation is shoot. Thirdly, rooting experiment was conducted by
developed to lay a technical foundation for its large-scale inoculating 2–3 cm long viable microshoots. The micro-
cultivation. shoots were harvested from the shooting media and were
transferred into culture bottles containing half strength
Materials and methods MS media. Six enriched rooting media (i.e., 0.75, 1.25,
Collection and sterilization of explants and 1.50 mg/L of NAA and 0.75, 1.25, and 1.50 IBA) and
Explants of A. elegans were collected from wild stands. one non-enriched media (control) were prepared in cul-
Collection of biological materials from the wild by native ture bottles in five replicates. One microshoot was cul-
(Ethiopian) researchers for research and development tured in each bottle and all microshoot-containing bottles
purposes is granted by Article 15, Clause 1 of the Access were incubated for four weeks. Rooting was operationally
to Genetic Resources and Community Knowledge, and defined as the emergence of ≤ 1 cm piece of root. Rooting
Community Rights Proclamation of Ethiopia (Proclama- shoots were kept in the growth room until many of the
tion No. 482/2006) without requiring written permission. shoots grow to ≥ 5 cm long plantlets. Initiation, shoot-
Specimen of the plant was identified by the second author ing, and rooting experiments were carried out in growth
and verified by a curator in the Aklilu Lemma Institute room racks at 25 ± 0.5 °C temperature under fluorescent
of Pathobiology, Addis Ababa University, Ethiopia; and a tube light, 16 h photoperiod, and 2,000–2,500 lx light
voucher specimen was deposited at the Endod and Other intensity.
Medicinal Plants Research Unit of the Institute (ID:
Welehaweria and Sbhatu BMC Research Notes (2023) 16:215 Page 3 of 7

Acclimatization study Results and discussion

Plantlets (46) with well-developed roots were harvested Initiation response
from the rooting media after four weeks; and were care- The initiation experiment resulted in 110 (78.6%) 1.5
fully washed with running tap water to remove any traces to 2.0 cm long clean and viable explants (i.e., initiated
of agar and sucrose. Then, they were soaked in hot water explants). All explants incubated in enriched media
(ca. 40 °C) for about 5 min to remove any oily stuff from were survived (100%) but only 5 out of the 35 (14.3%)
the root surface that would hamper water and nutrient plantlets incubated in unsupplemented media were sur-
absorption during acclimatization. Primary acclimati- vived. Many studies have succeeded in producing ini-
zation experiment was carried out in greenhouse. The tiated explants with offshoots using a combination of
plantlets were planted in Pro tray with cocopeat and kept different concentrations of BAP and NAA [7, 11–13]. In
in greenhouse for three-week primary acclimatization. vitro propagation media enriched with 0.20/0.20 mg/L,
The microclimate of the greenhouse was manipulated to 4.0/0.20 mg/L, 0.50/0.50 mg/L, and 0.20/0.20 mg/L BAP/
progress from high relative humidity (80–90), low tem- NAA have been effective for initiating growth in A. per-
perature (25 ± 2 °C), and low light intensity (1,200 lx) crassa Tod. [7], A. vera L. [11], A. trichosantha Berger
through medium relative humidity (70–80), medium [12], and A. adigratana Reynolds [13], respectively. BAP
temperature (26 ± 2 °C), and medium light intensity and NAA are often used for initiating explants of many
(2,500 lx) to low relative humidity (60–70), high temper- aloe species. However, establishing the right combina-
ature (27 ± 2 °C), and high light intensity (5,000 lx) over tions and concentrations for the best initiation response
the three weeks. Secondary acclimatization experiment requires further enquiry.
was conducted under nursery shade and direct sunlight.
Fourty one (41) plantlets with similar size and vigor that Shooting responses
survived primary acclimatization were divided into four Results of ANOVA indicated that the mean (±SD) num-
groups of 13, 10, 10 and 8 plantlets and transplanted into ber of days to shooting, shoot number, and shoot length
soil media. Two types of soil media composed of sand, were statistically significantly different among the treat-
soil, and compost (i.e., composted soil media) and sand, ments (p ≤ 0.05) (Fig. 1(a), (b) (c)).
soil, and manure (i.e., manured soil media) at 1:1:1 pro- Explants of A. elegans cultured in MS media enriched
portion were prepared and filled in polyethylene bags with different concentrations and combinations of BAP
(height 15 cm; diameter 9 cm). Two groups of plantlets and NAA took 13.0 ± 1.0 to 19.0 ± 2.0 days on aver-
were planted in the composted soil and the other two age to shoot. These results indicated that 13.0 ± 1.0 days
were planted in the manured soil. One group of plantlets to shooting (in MS medium enriched with 1.0 mg/L
in each soil medium was placed under nursery shade, and BAP + 0.50 mg/L NAA) was statistically significantly
the other group of plantlets was placed under direct sun- shorter (p ≤ 0.05) (Fig. 1(a)). Many other studies have
light. The plantlets were kept for three weeks by watering reported shooting in A. adigratana Reynolds, A. per-
daily with no nutritional supplements. crassa Tod., A. trichosantha Berger, and A. vera L. within
two weeks in MS media enriched with 1.0 mg/L BAP
Data collection and analyses in combination with auxins (such as IAA and NAA) [7,
Quantitative data including: number of explants surviv- 12–14].
ing initiation experiment, number of days to shoot and MS media enriched with BAP and NAA have pro-
root emergence, number of shoots and roots per bottle, duced 2.0 ± 1.0 to 5.0 ± 1.0 shoots within three weeks.
length of shoots and roots per plantlet, and survival rate Mean shoot number of 5.0 ± 1.0 per plantlet cultured in
of plantlets after acclimatization were recorded and orga- MS media enriched with 1.0 mg/L BAP + 0.25 mg/L NAA
nized for analyses. Data of days to shooting and rooting was significantly higher than the mean values observed
were collected by observing the incubated microshoots in other treatments (p ≤ 0.05) (Figs. 1(b) and 2). Combi-
every two days. Data on the number and length of shoots nation of BAP and NAA at 1.0 mg/L BAP and 0.50 mg/L
and roots were collected after the shooting and root- NAA, respectively, has resulted in significantly higher
ing experiments were concluded, respectively. Qualita- mean shoot numbers in many aloes species [7, 13, 15, 16].
tive observations were made to support the quantitative However, other studies have reported higher mean shoot
data. Data were analyzed through analysis of variance number per explant at higher concentrations of BAP [8,
(ANOVA) using the statistical package for social science 12, 17, 18].
(SPSS Version 20). Comparisons of mean (± SD) values Explants cultured in MS media supplemented with
were made at a priori set significance level of p ≤ 0.05. various concentrations and combinations of BAP and
NAA have produced shoots with mean length rang-
ing from 7.62 ± 1.60 cm (cultured in MS medium sup-
plemented with 2.0 mg/L BAP + 0.25 mg/L NAA) to
Welehaweria and Sbhatu BMC Research Notes (2023) 16:215 Page 4 of 7

Fig. 1 Different concentrations of BAP and constant concentration of NAA on shooting of A. elegans Tod

Fig. 2 Shooting response of A. elegans Tod. with different BAP/NAA supplementations. (Shoots in the smaller frame were cultured in MS medium with
1.0/0.25 mg/L BAP/NAA supplement)

9.20 ± 2.35 cm (cultured in MS medium supplemented High mean shoot lengths were also reported with lower
with 1.0 mg/L BAP + 0.50 mg/L NAA). The mean shoot (0.50 mg/L) [12, 19, 20] and higher (2.00 to 4.00 mg/L)
length of 9.20 ± 2.35 cm was significantly higher than the BAP in combination with 0.50 mg/L NAA [19–21].
rest of the mean values (p ≤ 0.05). No shooting response This research and so many other studies have gener-
was observed in the control (Fig. 1(c)). Many studies ally shown that the supplementation of low (0.50 mg/L)
with other species have reported the highest mean shoot through medium (2.0 mg/L) to high (4.0 mg/L) BAP in
length in plantlets cultured in MS media supplemented combination with 0.5 mg/L of NAA produce microshoots
with 1.0 mg/L BAP in combination with 0.5 mg/L NAA with high mean length. When all the variables of good
as compared to other combinations of PGR supplements shooting response are considered, 1.0–1.50 mg/L BAP
[7, 12, 13, 18]. in combination with 0.25–0.50 mg/L NAA lead to better
Welehaweria and Sbhatu BMC Research Notes (2023) 16:215 Page 5 of 7

micropropagation performance in Ethiopian aloes [7, 12, cultured in MS media enriched with 0.75 and 1.25 mg/L
13]. NAA produced 7.0 ± 4.0 roots per shoot (p ≤ 0.05). A
study with A. percrassa Tod. has resulted in higher mean
Rooting responses root number in shoots cultured in MS media with low
The ANOVA results showed that means of days to concentration of NAA supplements – decreasing with
rooting, number of roots, and length of roots were sta- increasing concentration from 8.4 in unsupplemented
tistically significantly different between the different medium to 3.4 with 1.50 mg/L NAA [7]. A similar trend
treatments (p ≤ 0.05) (Table 1). was observed in A. adigratana Reynolds [13]. Another
Microshoots grown in half-strength MS media sup- study on A. barbadensis has recorded the highest mean
plemented with three concentrations of NAA and IBA number of roots per shoot (4.8 ± 0.53) when cultured with
rooted in 12.00 ± 1.00 to 19.00 ± 2.00 days. Rooting 0.50 mg/L NAA supplementation [27]. Another study
medium supplemented with 0.75 mg/L IBA has resulted has also reported a better rooting response in A. indica
in rooting in 12.00 ± 1.00 days – statistically significantly L. with 0.50 mg/L NAA [26]. On the contrary, a research
less than all other treatments (p ≤ 0.05). Auxins (espe- with A. trichosantha Berger showed that the mean num-
cially IBA and NAA) are most commonly used PGRs ber of roots increases with increasing NAA supplements
for rooting of shoots [7, 12, 13, 16, 22–27]. Many stud- from 0.50 to 1.0 mg/L [12].
ies have reported rooting of aloe microshoots within one Similarly, the mean root length of shoots grown in
and three weeks in MS media supplemented with 1.0 to MS media supplemented with NAA and IBA ranged
2.0 mg/L of IBA [7, 13, 25]. Similarly, rooting media sup- from 3.62 ± 2.37 to 7.53 ± 3.03 cm. Higher IBA con-
plemented with 0.50–1.50 mg/L NAA have caused root- centrations have resulted in higher mean root lengths
ing responses in less than 15 days [7, 12, 13, 22]. Many (Table 1). On the other hand, higher mean root lengths
studies have shown that aloe shoots develop roots in were observed with increasing concentration of NAA
9–30 days but it is hard to draw clear pattern when PGRs’ from 0.75 to 1.50 mg/L. In a study with A. adigratana
concentrations increase or decrease [7, 12, 13]. Reynolds, mean root length decreased with increasing
The mean root number of the rooted shoots, tech- NAA concentration from 0.50 to 1.50 mg/L while the
nically known as plantlets, ranged from 4.00 ± 2.00 to opposite was observed with IBA [13]. In one study with
8.00 ± 4.00. Half-strength MS medium enriched with A. trichosantha Berger, it was shown that the mean root
0.75 mg/L IBA has produced plantlets with significantly length decreases as NAA concentration decreases from
higher mean root number (8.00 ± 4.00; p ≤ 0.05) (Table 1). 0.25 to 1.50 mg/L [12]. Other researchers have observed
A. adigratana Reynolds shoots cultured in rooting media similar patterns with unsupplemented and low NAA
enriched with 0.50 to 1.50 mg/L IBA have produced 8.4 supplemented (0.50 mg/L) media producing the high-
to 11 roots per shoot [13]. IBA (1.0 mg/L) in combination est mean root lengths of up to 6.0–7.0 cm [7, 11, 28]. In
with activated charcoal (500 mg/L) has resulted in higher the future, optimization of rooting media for A. elegans
mean root number (5.42) per explant in A. barbadensis shoot culture should focus on lower NAA and higher IBA
Mill. [17]. The present study has also shown that shoots supplementations.

Table 1 Different concentrations of NAA and IBA on rooting Acclimatization

responses of A. elegans Tod Fourty five (97.8%) of A. elegans plantlets exposed to pri-
PGRs Con- Mean (SD) values mary acclimatization in the greenhouse have survived.
centra- No. of Root Root Many studies on aloes have reported high primary accli-
tion days to number length, matization rates – e.g., 100% with A. trichosantha Berger
(mg/L) rooting cm [12], 96.6–100% with A. adigratana Reynolds [13],
Control 0.00 – – – 94–100% with A. percrassa Tod. [7], 95–100% with A.
NAA 0.75 17.0 (3.0)a 7.0 (4.0)b 5.2 (2.3)b vera L. [14, 18], and 95% with A. barbadensis Mill. [17].
1.25 17.0 (2.0)a 7.0 (4.0)b 3.8 (2.2)a Acclimatization media affect the success of acclimatiza-
1.50 17.0 (3.0)a 4.0 (3.0)a 3.6 (2.4)a tion of the delicate heterotrophic plantlets. Coco peat,
IBA 0.75 12.0 (1.0)b 8.0 (4.0)c 4.4 (2.0)a composted and manured soil, perlite and vermiculite
1.25 19.0 (2.0)a 4.0 (2.0)a 7.5 (3.0)c soil, and other media are preferred because they facilitate
1.50 17.0 (1.0)a 7.0 (4.0)b 7.4 (2.2)c
drainage and aeration. Primary and secondary acclimati-
Mean 14.72 5.48 4.14
zation trials with coco peat often yield up to 100% sur-
CV% 45.00 76.00 72.00
vival rate [7, 12, 13].
LSD 0.07 0.00 0.03
The secondary acclimatization experiment has yielded
Means in the same column with different letters are statistically significantly
different at p ≤ 0.05; CV: Coefficient of variance (%); LSD: Least significant 87.5 to 92.3% survival rate; with a single plantlet died
different from each group. Aloes are hardy plants capable of
Welehaweria and Sbhatu BMC Research Notes (2023) 16:215 Page 6 of 7

Acknowledgements
withstanding harsh environmental conditions. It is, thus, The authors acknowledge the Tigrai Biotechnology Center Pvt. Ltd. Co. for
natural for aloe species to easily acclimatize. The pres- allowing us to carry out the laboratory and greenhouse studies.
ent study has observed similar survival rates of plantlets
Authors’ contributions
subjected to secondary acclimatization with no differ- D.B.S. and M.W. were involved in conceiving, designing, and planning of the
ence due to rooting media or microclimate. The second- study; D.B.S. carried out the identification and field location of the plant; M.W.
ary acclimatization tests under shaded nursery or direct carried out the collection of the plant materials, the experimentation, data
collection and analyses, and draft manuscript write up, and D.B.S. reviewed
sunlight did not make a difference. We have observed the manuscript for content, language, and style; and prepared the manuscript
that the death of the single plantlet in each treatment was for publication.
not linked to physiologic or anatomic reasons but due to
Funding
physical damages. Regardless of the planting soil media This study was supported by Mekelle University, PO Box 231, Mekelle, Ethiopia
or light conditions (nursery shade or direct sunlight), through the Grant No.: CRPO/MIT/LARGE/001/09.
high survival of aloe plantlets subjected to secondary
Data Availability
acclimatization is common [7, 12–14, 18]. Healthy and The datasets used and/or analyzed during the current study are available from
undamaged plantlets often survive and grow profusely. the corresponding author on reasonable request.

Conclusion Declarations
This study has shown that initiation and shooting of
Ethics approval and consent to participate
A. elegans explants required PGRs-supplemented MS Not applicable.
media. Explants of the species responded to full-strength
MS shooting media enriched with 1.0/0.50 mg/L BAP/ Consent for publication
Not applicable.
NAA in less than two weeks. MS shooting media supple-
mented with 1.0 mg/L BAP in combination with 0.25 to Competing interests
0.50 mg/L NAA were effective in yielding the highest No competing interests to disclose.

mean shoot number and length. Likewise, well-developed

Received: 6 March 2023 / Accepted: 1 September 2023
shoots of this aloe produced roots in less than two weeks
in half-strength MS media enriched with 0.75 mg/L IBA.
IBA supplementation of rooting media at 0.75 mg/L has
caused the highest mean number and length of roots.
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