Al-Nema and Abdullah

RESEARCH ARTICLE(2023)

SABRAO Journal of Breeding and Genetics

55 (1) 254-259, 2023
http://doi.org/10.54910/sabrao2023.55.1.23
http://sabraojournal.org/
pISSN 1029-7073; eISSN 2224-8978

PROPAGATION PROTOCOL OF THE MEDICINAL PLANT - ALOE VERA USING TISSUE

CULTURE

Q.S. AL-NEMA* and R.M. ABDULLAH

Department of Biology, College Education for Pure Sciences, University of Mosul, Mosul, Iraq
*Corresponding author’s emails: dr.qutaibashuaib@uomosul.edu.iq
Email address of co-author: raghad.mohammed@uomosul.edu.iq

SUMMARY

Aloe vera is one of the most popular cactus-type plants in the global market due to its widespread
uses in pharmaceutical, cosmetic, food, and decorative purposes. The present study derived callus
cultures from the Aloe vera plant leaves, then reproduced on agar-solidified MS medium from June to
December 2021 at the University of Mosul, Iraq. Results revealed that the MS medium + 3.0 mg L -1
benzyl adenine (BA) proved suitable for induction of leaf callus up to 85%, while the MS medium
supplement with 1.0 and 2.0 mg L-1 BA reached 70%. The MS medium with 1.0 mg L -1 BA showed the
best results for growing apical shoots of A. vera plants and producing vegetative branches. The
formation of roots emerged within two weeks after placing them on the rooting medium. The shoots
regenerated from the growing apices and were rooted easily in agar-solidified MS medium. The
obtained plants attained successful acclimatization in terms of their growth and length, afterward,
transferred to the peat-moss mixture.

Keywords: Aloe vera, callus cultures, propagation, leaves, stems, apical shoot

Key finding: The study aimed to identify the behavior of Aloe vera plants in the culture medium
represented by the formation of callus cultures and their differentiation.

Communicating Editor: Prof. Naqib Ullah Khan

Manuscript received: December 21, 2022; Accepted: January 20, 2023.

© Society for the Advancement of Breeding Research in Asia and Oceania (SABRAO) 2023

INTRODUCTION off easily for immediate placing on cuts and

burns for relief.
Aloe, a popular indoor house plant, has a long Aloe vera is a clump-forming succulent
history as a multipurpose folk remedy, whose fleshy gray-green leaves form in a
commonly known as Aloe vera, native to Africa rosette shape atop a very short stem. The
and Spain (Sajjad and Sajjad, 2014). Aloe Vera leaves have small grayish teeth on the
is a member of the family Liliaceae and has margins. The central rosette gets up to about
cactus-like characteristics. The plant has two feet high, and the plant continuously
several therapeutic and pharmacological produces little offset rosettes. In winter and
properties, as well as, possesses spring, medicinal aloe bears small tubular
counteractions for oxidation (Rajeshwari and yellow flowers on inflorescence up to three feet
Andallu, 2011). Aloe vera is a shrubby plant tall. The original Aloe vera has yellow flowers,
with many medicinal applications and but most available varieties have orange
substantial uses in cosmetics and health care flowers. The human body requires 22 amino
(Eshun and He, 2004). The plant stem snaps acids for good health, of which eight consist

Citation: Al-Nema QS, Abdullah RM (2023). Propagation protocol of the medicinal plant - aloe vera using tissue
culture. SABRAO J. Breed. Genet. 55(1): 254-259. http://doi.org/10.54910/sabrao2023.55.1.23.

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 Al-Nema and Abdullah (2023)

"essential amino acids" because the body 2010). Therefore, this research is a vital step
cannot fabricate them. Aloe vera contains over in plant biotechnology to preserve the essential
20 minerals that are essential to the human genotypes of medicinal plants, providing great
body. Aloe Vera also contains eight essential potential for producing high-quality plant
amino acids and 11 of the 14 "secondary medicines. The current study aimed to identify
amino acids." Aloe vera also has vitamins A, the response of Aloe vera plants in the culture
B1, B2, B6, B12, C, and E (Surjushe et al., medium represented by the formation of callus
2008). cultures and their differentiation.
Conventional plant breeding methods
can improve both agronomic and medicinal
traits. In vitro propagation or tissue culture of MATERIALS AND METHODS
plants holds tremendous potential for
producing high-quality plant-based medicines. Explant source
Different methods can achieve this, including
micropropagation (Yushkova et al., 1998; One-year-old Aloe vera plants obtained from
Jathunarachchi et al., 2021; Almukhtar, 2022). private nurseries in Mosul City, Iraq,
Plant tissue culture is one of the significant underwent thorough washing with tap water
approaches for producing healthy plants free and detergent (liquid soap) for 10 min.
from diseases, especially viral ones. This Carefully separating explants ranged with 3–6
technology has provided broad horizons for leaves and apical shoot, their surfaces gained
improving plant species of economic and sterilization with 3% NaOCl (sodium
medical importance (Cardoza, 2008). The use hypochlorite) for 5 min, afterward, washed 3–4
of tissue culture technique has obtained times with sterile autoclaved distilled water to
numerous plants speedily to conserve the remove NaOCl (Chukwujekwu et al., 2002).
genetic characteristics of various plant Distributing 20 ml agar-solidified MS medium
resources (Purohit, 2007). proceeded into flasks with 250 ml capacity.
Several studies indicated the possibility Upon approval, the current research work took
of propagating A. vera through tissue culture place from June to December 2021 following
to produce callus cultures from leaves on agar- the instructions of the Central Scientific
solidified MS medium, supplemented with the Research Ethics Committee at the University of
addition of 0.5 mg L-1 of each 2,4-D and Kin, Mosul, Iraq.
with shoot regeneration by the addition of 1.0
mg L-1 BAP and 0.5 mg L-1 NAA (Murashige and Callus induction
Skoog, 1962; Choudhary et al., 2011). Another
recent study indicated that Aloe vera The growth regulators, such as 2,4-D and BA,
responded best in vitro growth in leaf explants were added and tested in concentrations of
cultivated on MS medium supplemented with 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg L-1 to the
1.0 mg L-1 BAP and 1.0 mg L-1 NAA (Wahab et MS medium. Aloe vera leaf explants cut into
al., 2020). portions of 1.0 cm2 received each treatment,
Enhancing the production with the including 10 flasks with 2.0 pieces of leaf
maximum number of shoots of A. vera plants explants per flask. The samples placed at 25
resulted from agar-solidified MS medium °C temperature underwent alternate light
provided with a mixture of 1.0 mg L-1 IAA and conditions of 16 h of light / 8 h of darkness.
4.0 mg L-1 BAP. The same MS medium
supplemented with 0.2 mg L-1 IAA and 0.8 mg Culture of apical shoot
L-1 BAP achieved the rooting of regenerated
shoots in the same medium composition Sterilized apical shoot and stems (length 2.0
(Molsaghi et al., 2014). Reports also stated the cm) of Aloe vera followed cultivation by
high shoot production at 2.90, and the implanting their bases upright in 25 ml of MS
multiplication factor approached 2.87 on MS medium supplemented each with 1.0, 1.5, 2.0,
medium supplemented with 0.2 mg L-1 IBA and and 2.5 mg L-1 BA (Chukwujekwu et al., 2002),
1.0 mg L-1 BAP, respectively (Surafel et al., planting their bases in the center with one tip
2018). per flask. Samples remained in the culture
As it is known, the Aloe vera plant room in the same condition previously
reproduces naturally by offshoots, with a mentioned. Cutting out Aloe vera vegetative
relatively slow way of multiplication to meet branches obtained from cultivation of the
the growing demand. Each plant produces 2–3 apical shoot and stems used a sharp scalpel,
offshoots per year, showing insufficient for with their bases planted upright in 25 ml of
commercial cultivation (Saggoo and Kaur,

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agar-solidified MS medium supplemented with medium containing 2,4-D (Figure 1-A), while
1.0 mg L-1 NAA (Hailu et al., 2020). the callus appeared white in the MS medium
Regenerated parts of Aloe vera containing BA (Figure 1-B).
removed from the culture medium, and roots The results specified that apical shoot
proceeded water-rinsing to remove the cultivation produced intact plants readily, later
medium. Planting a group of these plants in cultivated in various culture media. The
peat moss took place, with another group investigations also showed a significant
planted in a mixture of soil and peat moss in decrease in the number of shoots obtained
plastic pots, then covered with transparent and with growing apical shoots with increased BA
perforated nylon bags for seven days. Later, concentration in the MS medium (Table 2).
removing the bags allowed the plants to However, it does not encourage the formation
continue growing in the cultivation room under of shoots from stem explant in said MS
the earlier mentioned conditions. medium.
It also seemed that apical shoots
showed to regenerate many shoots on agar-
RESULTS solidified MS medium (Figure 1-C). Meanwhile,
the agar-solidified MS medium containing 1.5
The results indicated that, in general, mg L-1 BA regenerated several vegetative
difficulties also occurred during the shoots ranging between 3–5 branches (Figure
development of calli from Aloe vera leaves, and 1-D). The results also exhibited that the MS
the MS medium provided with the addition of medium with 1.0 mg L-1BA provided the best
3.0 mg L-1 benzyl adenine (BA) demonstrated medium for producing vegetative twigs from
superior for leaf callus development, with the stem explants (Figure 1-E). However, all the
recorded development rate at 85% (Table 1). treatments required three to eight weeks to
However, observations showed that the Aloe make the vegetative branches in good
vera samples planted on other media did not condition (Table 2).
encourage development significantly. The vegetative shoots produced from
Results further indicated that leaf the apical shoot rooted fast, with their bases
explants involved 12–20 days to start the cultivated in agar-solidified MS medium
callus development. The results showed that supplemented with 1.0 mg L-1 NAA. The
few explants emerged with enhanced induction formation of roots appeared within two weeks
of callus from the edges of the explant; of placing them on the rooting medium (Figure
however, it also stopped soon after a short 1-F). The results also displayed vegetative
period. The callus texture was compact and shoots that showed success after transferring
yellowish-green in color grown in the MS to the peat moss, then acclimatized in terms of

Table 1. Callus induction from leave explant of Aloe vera plant.

Induction media (mg L-1) Total number of induced explants* Induction (%)
MS (control) 0 0
MS + 0.5 BA 16 80
MS + 1.0 BA 14 70
MS + 1.5 BA 15 75
MS + 2.0 BA 14 70
MS + 2.5 BA 16 80
MS + 3.0 BA 17 85
MS + 0.5 2,4-D 0 0.0
MS + 1.0 2,4-D 2 10
MS + 1.5 2,4-D 0 0.0
MS + 2.0 2,4-D 4 20
MS + 2.5 2,4-D 0 0.0
MS + 3.0 2,4-D 0 0.0
MS + 0.5 2,4-D + 1.0 BA 10 50
MS + 1.0 2,4-D + 1.0 BA 8 40
MS + 1.5 2,4-D + 1.0 BA 2 10
MS + 2.0 2,4-D + 1.0 BA 11 55
MS + 2.5 2,4-D + 1.0 BA 13 65
MS + 3.0 2,4-D + 1.0 BA 11 55
*20 samples per treatment

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Figure 1. Plant propagation and callus formation from the stem and apical shoot explant of Aloe vera
L.
(A) Leaf callus on MS medium + 2.0 mg L-1 2,4-D (15 days age). (B) Leaf callus on MS medium + 3.0 mg L-1 BA
(18 days age). (C) Formation of shoots after three weeks from the apical shoot on MSO medium. (D) Formation of
shoots after four weeks from the apical shoot on MS medium + 1.5 mg L-1 BA; note the number of branches
(arrows). (E) Formation of shoots after four weeks from stems on MS medium + 1.0 mg L -1 BA. (F) Rooting of
shoots resulting from cultivation of apical shoot on MS medium + 1.0 mg L-1 NAA. (G) Acclimatization of plantlet
produced from the cultivation of apical shoots in peat moss. (H) Increasing growth of plants and formation of
additional leaves after three weeks of acclimatization.

Table 2. Formation of vegetative shoots of Aloe vera from the apical shoot and stem explant grown
on differentiation media.
Differentiation media Total number of explants
Explant Induction (%) Time (week)
(mgl-1) Tested Induced
Apical MS + 0.0 BA 10 7 70 4
shoots MS + 1.0 BA 10 5 50 3
MS + 1.5 BA 10 3 30 7
MS + 2.0 BA 10 2 20 4
MS + 2.5 BA 10 0 0.0 0
Stems MS + 0.0 BA 10 0 0.0 0
MS + 1.0 BA 10 4 40 8
MS + 1.5 BA 10 0 0.0 0
MS + 2.0 BA 10 0 0.0 0
MS + 2.5 BA 10 2 20 6

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enhanced growth and length reaching 5–7 cm attribute to the fact that the level of
(Figure 1-G). However, seven of the total endogenous auxins was high enough to
plants observed have continuous development, promote the said process in the species Aloe
producing additional leaves (Figure 1-H). polyphylla (Chukwujekwu et al., 2002).
Several reports have noted rapid in
vitro propagation of A. vera (Aggarwal and
DISCUSSION Barna, 2004; Hosseini and Parsa, 2007).
Scientists obtained different results applying a
Several past studies on the Aloe vera plant different formulation of plant growth regulators
concentrated on propagation using stems and for in vitro propagation of A. vera. The
shoot tips, rooted easily with the formation of hormonal requirement for in vitro
whole plants (Hailu et al., 2020). It seems differentiation differs for different genotypes.
likely that certain obstacles faced by callus The ability of plants to stimulate callus and
formation than the complexities challenged its produce vegetative branches depends on the
growth histologically, despite providing the internal hormonal level, and growth regulators
required media and supporting it with added to the nutrient media until they reach
reasonable growth regulator types and the equilibrium stage (Acharjee et al., 2012).
concentrations. It could refer to the Aloe vera Several researchers have reported different
plant containing a group of chemical responses to plant production with A. vera.
compounds that may sometimes hinder the Daneshvar et al. (2013) noted that the highest
differentiation processes (Surjushe et al., plant yield obtained used 0.15/2.5 mg/l
2008). BAP/NAA. Zakia et al. (2013) also recorded
Growth regulators could play a vital that the tallest vegetative shoot formation
role and manifest themselves in different ways emerged using 0.5/0.5 mg L-1 BAP/NAA.
in rebellious plant species. Studies indicate Moreover, other researchers observed 14 to 16
that BAP and IBA had an effective and shoots per plant with 4.0/0.2 mg L-1 of
significant role in plant organogenesis by direct BAP/NAA in A. vera (Nayanakantha et al.,
differentiation of shoots from cotyledon leaf 2010; Khanam and Sharma, 2014). This
explants in Catharanthus roseus (Abdul-Jaleel difference could attribute to the type of plant
et al., 2009). In the Tagetes erecta plant, and genetic variation that often affect the
multiple shoots resulted with the MS medium response of tissue culture (Lobine et al.,
containing BAP, with the plant growth 2015).
regulators (PGRs) added to the media acting as
stimulators of metabolic pathways for
phytochemicals. Therefore, the regenerated CONCLUSIONS
plants appeared metabolically similar or even
superior to the parental plant (Misra and Datta, Establishing a successful protocol for creating
2001). The type of plant part used in Aloe vera plants from stems and apical shoot
micropropagation proved to have an effective constitutes an imperative path in producing
role in the formation of vegetative branches, that crucial medicinal plant by tissue culture.
indicating more success and efficiency in the
axillary buds in the propagation of A. vera
(Molsaghi et al., 2014). Other researchers also ACKNOWLEDGMENTS
recommend using the tip of the plant and the
apical tissue for micropropagation of A. vera The authors gratefully thank the College of Education
(Ahmed et al., 2007). for Pure Sciences, University of Mosul, Mosul, Iraq,
Several studies described the process for providing the facilities required for the present
investigations.
of callus formation and differentiation in Aloe
vera as a multipurpose plant with medicinal
values (Nayanakantha et al., 2010). Cultivation
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