Medicinal plants are in use in many countries and cultures as a source of medicine. Biotechnological tools like tissue culture are important for selection, multiplication and conservation of medicinal plants genotypes. In addition, in-vitro regeneration plays a great role in the production of high-quality plant-based medicine. Plant tissue culture techniques offer an integrated approach for the production of standardized quality phytopharmaceutical through mass production of consistent plant material for physiological characterization and analysis of active ingredients. A number of medicinal plants reported to regenerate in vitro from their various parts but still, fewer are grown in soil, while their micropropagation on a mass scale has rarely been achieved. Micropropagation protocols for cloning of some medicinal plants had been developed by using different concentrations of plant growth regulators in a Murashige and Skoog media variant (Murashige and Skoog, 1962). Regeneration occurred via organogenesis and embryogenesis in response to auxins and cytokinins. The production of secondary metabolite is also becoming familiar by tissue culture for pharmaceutical use. The integrated approaches of culture systems will provide the basis for the future development of safe, effective, and high-quality products for consumers.

1. Micropropagation of Medicinal Plants: Review
Micropropagation of Medicinal Plants: Review
*Dadi Tolessa Lemma1, Dejene Tadesse Banjaw2 and Habtamu Gudisa Megersa3
1,2,3Ethiopia Institute of Agricultural Research, Wondo Genet Agricultural Research Center, P.o.box 198, Shashemene,
Ethiopia
Medicinal plants are in use in many countries and cultures as a source of medicine.
Biotechnological tools like tissue culture are important for selection, multiplication and
conservation of medicinal plants genotypes. In addition, in-vitro regeneration plays a great role
in the production of high-quality plant-based medicine. Plant tissue culture techniques offer an
integrated approach for the production of standardized quality phytopharmaceutical through
mass production of consistent plant material for physiological characterization and analysis of
active ingredients. A number of medicinal plants reported to regenerate in vitro from their various
parts but still, fewer are grown in soil, while their micropropagation on a mass scale has rarely
been achieved. Micropropagation protocols for cloning of some medicinal plants had been
developed by using different concentrations of plant growth regulators in a Murashige and Skoog
media variant (Murashige and Skoog, 1962). Regeneration occurred via organogenesis and
embryogenesis in response to auxins and cytokinins. The production of secondary metabolite is
also becoming familiar by tissue culture for pharmaceutical use. The integrated approaches of
culture systems will provide the basis for the future development of safe, effective, and high-
quality products for consumers.
Key words: BAP, In vitro propagation, regeneration, secondary metabolites, somatic embryogenesis, organogenesis
INTRODUCTION
Most of plant species were used as source of medicine for
many years. Medicinal plants used almost in all countries
and cultures as a source of medicine. Medicinal plants
have important role in health care systems (Denbath et al.,
2006). World health organization estimated that herbal
medicines serve the health needs about 80% of the world’s
population, especially for millions of people in rural areas
of developing nations. Medicinal plants are also the source
for many modern medicines. According to some reports
one quarter of legal drugs contain plant extracts or
modeled on plant substances. The most popular
analgesic, aspirin, was originally derived from species of
Salix and Spiraea and some of the most valuable anti-
cancer agents such as paclitaxel and vinblastine are
derived solely from plant sources (Pezzuto, 1996).
High amount of herbal medicine is used in developed
country. Around 25% population in UK takes herbal
medicines in most cases for disease treatments. Almost
two thirds of the 50,000 different medicinal plant species
are collected from wild. In Europe, only 10% of medicinal
species used commercially are cultivated. Currently there
is a worry about decreasing populations, loss of genetic
diversity, local extinctions and habitat degradation of
medicinal plants. Some reports indicated that more than
4,000 medicinal plant species might now be endangered
(Goel and Singh, 2009).
The market for plant-derived chemicals in
pharmaceuticals, fragrances, flavors, and color
ingredients, alone exceeds several billion dollars per year.
For example, plant-based substances like taxol,
vincristine, vinblastine, colchicine, artemisinin, forkolin,
Saponin are highly used in biology and medicines.
Medicinal plants growing and exporting is increasing in
world trade market. The herbal market is growing by the
rate of seven percent which is $60-billion currently is
expected to generate more than $5 trillion by 2050
(Denbath et al., 2006).
*Corresponding Author: Dadi Tolessa Lemma Ethiopia
Institute of Agricultural Research, Wondo Genet
Agricultural Research Center, P.o.box 198, Shashemene,
Ethiopia. Email: daditolessa2003@gmail.com
Co-Author 2
Email: dejenebangaw@gmail.com
3
Email: habtegudisa21@gmail.com
International Journal of Plant Breeding and Crop Science
Vol. 7(2), pp. 796-802, September, 2020. © www.premierpublishers.org, ISSN: 2167-
0449
Review Article

2. Micropropagation of Medicinal Plants: Review
Lemma et al. 797
The production and consumption as well as international
trade in medicinal plants, and phytomedicines, are growing
quite significantly. In addition, consumption of herbal
medicines is widely spread and increasing. However,
harvesting of herbal medicine from the wild as a source of
raw material is causing loss of genetic diversity and habitat
destruction. In spite of herbal treatments for curing
different ailments from the ancient times, even today large
numbers of medicinal plants are being harvested from their
wild habitat (Kumari and Priya, 2020). Because there is no
control over their harvesting, so the agents of traders and
Vaidaya, do harvest them mercilessly and due to this
several species have become extinct or are on the verge
of extinction (Kumari and Priya, 2020). Plant tissue culture
techniques are said to be more suitable alternative to help
in this alarming problem. Establishment of cell, tissue and
organ culture and regeneration of plantlets under in vitro
conditions has opened up new avenues in the areas of
plant biotechnology (Dagla, 2012). Micropropagation is the
process of vegetative growth and multiplication from viable
and regenerative cells in aseptic and favorable condition
on suitable culture medium using various plant tissue
culture techniques (Zhou and Wu, 2006). Because through
in vitro propagation large numbers of identical plants can
be produced within a limited space and time, which can be
used as planting materials, this technique is being used in
the Micropropagation of different medicinal plants.
Application of traditional and biotechnological plant-
breeding techniques used to improve the genetic level for
improving yield and uniformity. In vitro propagation or
tissue culture of plants is very important for the production
of high-quality plant-based medicines. This can be
achieved through different methods including
micropropagation (Yushkova, 1998). The evolving
commercial importance of secondary metabolites in recent
years resulted in a great interest in secondary metabolism,
particularly in the possibility of altering the production of
bioactive plant metabolites using tissue culture
technology. Many cell culture technologies introduced at
the end of the 1960's as a possible tool for both studying
and producing plant secondary metabolites (Tripathi and
Tripathi, 2003). Different in vitro systems, has been to
improve the production of plant chemicals.
In vitro plant culture is an important technique for mass
multiplication of plants, elimination of plant diseases
through meristematic tissue culture technique, plant
conservation and crop improvement through gene transfer
(Sarasan et al., 2011). Therefore, prevention of
contamination from different sources like bacteria and
fungi is necessary for successful culture of medicinal
plants by using in vitro propagation.
As most of the plants are not cultivated or micropropagated
under similar conditions, they vary in their characteristics.
Secondary metabolites vary from season to season and
developmental stage of the plant. Sustainable production
of drugs in the pharmaceutical industry depends on
continuous supply of healthy material to which plants
provide a major contribution (Sahoo et al., 1997).
Developing reliable propagation protocols of these
economically important medicinal plants through
micropropagation is very important for rapid regeneration
and quality planting materials for production.
Explants source and sterilization
Materials used for tissue culture propagation is known as
explants. Success tissue culture mainly depends on the
age, types and position of explants (Gamborg et al., 1976)
because all plans have not equal totipotency. Shoot tips,
nodal buds and root tips are the commonly used source of
explants. Large explants can increase chances of
contamination and small explants like meristems can
sometimes show less growth (Murashige and Skoog,
1962).
Sterilization of explants is one of the major steps for
successful in vitro micropropagation. Agents like calcium
hypochlorite, sodium hypochlorite, ethanol, mercuric
chloride, hydrogen peroxide, or silver nitrate are for
sterilization (Mihaljevic et al., 2013). Sterilization is used to
reduce the contamination and to get disease-free explants.
The selection of sterilizing agent depends on the type of
explants depending on the morphological characteristics
like hardness and softness of the tissue (Yadav and Singh,
2011b).
Microbes multiply and compete with growing explants for
nutrients, while releasing chemicals, which can alter
culture environments e.g. pH can inhibit explants growth
or cause death (Leifert and Waites, 1992). Explants
cleaned by distilled water and sterilized using mercuric
chloride, ethyl alcohol, and liquid bleach (Matkowski,
2008). Sterilization of laboratory instruments carried out by
autoclaving, alcohol washing, baking, radiations, flaming
and fumigation. A considerable decrease in bacterial
contamination was seen by using an ultrasonic sonicator
(Monge et al., 2008).
The beginning of micropropagation of medicinal
plants
Micropropagation of medicinal plants remained neglected
until complete plants of Rauvolfia serpentine L. were
produced from its somatic callus tissue (Mitra and
Chaturvedi, 1970). The performance of tissue-cultured
plants depends on the selection of the initial material,
media composition, growth regulators, cultivar and
environmental factors. The effects of auxins and cytokinins
on shoot multiplication of various medicinal plants has
been reported. Skirvin et al (1994) observed a rapid
proliferation rate in Picrorhiza kurroa using kinetin at 1.0–
5.0 mg/l. Barna and Wakhlu (1998) has indicated that the
production of multiple shoots is higher in Plantago ovata
on a medium having kinetin along with NAA. Faria and Illg
(1995) have also shown that the number of shoots per

3. Micropropagation of Medicinal Plants: Review
Int. J. Plant Breed. Crop Sci. 798
explant depends on concentrations of the growth
regulators and the particular genotypes. The nature and
condition of explants also have a significant influence on
the multiplication rate. Mao et al. (1995) reported that the
actively growing materials were more responsive to shoot
induction than dormant buds in Clerodendrum
colebrookianum.
Regeneration and organogenesis
Micropropagation is among the most commercially
efficient and practical plant propagation technologies. In-
vitro propagation methods rely on the totipotency of plant
cells. Direct organogenesis is generally considered the
safer route for micropropagation of clonal, true-to-type
plants (Sandhu et al, 2018); this complex process involves
synergistic interactions between physical and chemical
factors (Chand et al., 1997) and is initiated within the shoot
(or root) meristem of an explant (Altman and Loberant,
1998). This is mainly controlled by the endogenous and
exogenous balance of plant growth regulators. Genotype,
explant type, and physiological status modulate
endogenous levels of these regulators. In particular, the
endogenous levels of these regulators are influenced by
the composition of the culture medium (especially the
concentration and type of plant growth regulators (PGRs),
which in turn greatly influences the direction and efficiency
of organogenesis (Amer and Omar, 2019).
Plant regeneration and development are regulated by
plant hormones; indeed, most physiological processes
involve the interplay of several phytohormones (Wang and
Irving, 2001) that act synergistically or antagonistically
(Gaspar et al., 2000). As a result, the use of multiple
hormones versus a single hormone is more effective, in
most cases, for plant regeneration.
In organogenesis, the apical meristem of shoot apex,
axillary buds, root tips, and floral buds are stimulated to
differentiate and grow into shoots and finally into complete
plants. The explants cultured on relatively high amounts of
auxin form an unorganized mass of cells, called callus.
Differentiation and organogenesis accomplished from
callus by using different growth regulators in culture
medium. Endogenous growth substances or addition of
exogenous growth regulators to the nutrient medium
stimulate cell division, cell growth and tissue
differentiation. There are many reports on the regeneration
of different medicinal plants via callus culture. According to
Pande et al (2002), the successful in-vitro regeneration of
Lepidium sativum from various explants on MS (Murashige
and Skoog) medium, which is, supplemented with 4.0 mg/l
BAP and NAA. The role of auxins and cytokinins in callus
induction was also advocated by Patel and Shah (2009) in
Stevia rebaudiana, through callus culture. They have
standardized callus induction and multiplication medium
from nodal as well as leaf segments. For callus induction,
explants were cultured on MS medium, with varying
concentration of BA and NAA. 2.0 mg/L BA + 2.0 mg/L
NAA is best for callus induction, and higher regeneration
frequency was noticed with this combination. Protocol
standardization done for Rauwolfia serpentina from shoot
tip culture showed best response for shoot proliferation
was observed in MS medium containing 0.1 mg/L NAA and
2.5 mg/L BA, where 92% of plants showed proliferation.
For rooting, half- strength MS medium supplemented with
0.4 mg/L NAA and 0.1 mg/L IBA showed maximum root
formation (Susila et al., 2011). Yadav and Singh (2010)
reported that maximum possibility of adventitious roots
induction was induced from middle node apex for
Spilanthes acmella showed good result on full-strength MS
medium supplemented with 1.0 mg/l BAP under the
photoperiod of 18-h. The possibility of adventitious roots
induction directly from regenerated shoot was greatly
influenced by the concentration of BAP, photoperiod, age
of donor plant and nodal position on stem. Baishya et al.,
(2015) reported direct shoot regeneration of A. annua L.
using leaf explants on MS medium supplemented with
BAP (3mg/l) and 1/2MS+ IBA (3mg/l) resulting in a rapid
and high number of shoots per explants.
Callus induction from nodal explants of Periwinkle
(Catharanthus roseus) was observed on Murashige and
Skoog (MS) medium supplemented with NAA (0.2 mg/l)
and KN (2mg/l). Multiple shoot proliferation and shoot
elongation were observed on MS medium supplemented
with NAA (0.5mg/l) and KN (2mg/l). These shoots when
transferred to MS medium supplemented with IBA (2mg/l)
resulted in rooting (Debnath et al., 2006). In vitro
micropropagation developed for Piper crocatum on MS
culture medium containing 5.0 mg/L BAP +0.5 mg/L 2,4-D
that supplemented with activated charcoal gave the most
suitable media for shoot initiation with less browning
problem (Zuraida et al., 2015). Shoot regenerated from
callus MS medium with 2.5 mg/l BA and 0.1 mg/l NAA and
half strength MS medium with 0.1 mg/l NAA is optimal for
rooting of Pelargonium graveolens (Gupta et al., 2002).
Tissue culture studies for in vitro multiple shoots induction
in medicinal plants have been done by Gupta et al., (2001)
in Lippia alba., Pan et al., (2003) in Artemisia and Echinops
spp. Bhavisha and Jasrai (2010) reported that maximum
number of roots were induced in plantlets of Curculigo
orchioides raised through tissue culture in lowest. 1.0 mg/l
concentrations of NAA. Whereas Sasikumar et al., (2009)
reported that thick and long roots were induced in plantlets
of Baliospermum montanum in MS + 1.0 mg/l IBA and 0.5
mg/l IAA. Also, some scholars reported that maximum
number of shoots on nodal explants of Phyla nodiflora than
the shoot tip explants have been reported by Ahmad et al.,
(2010).
Somatic Embryogenesis
Somatic embryogenesis is an important technique for plant
regeneration. This technique has been used for plants like
date palm plant regeneration from embryogenic culture
(Abohatem et al., 2017). Somatic cells or tissues lead to

4. Micropropagation of Medicinal Plants: Review
Lemma et al. 799
the formation of somatic embryos, which look like the
zygotic embryos of intact seeds and can grow into
seedlings on suitable medium. Plant regeneration via
somatic embryogenesis from single cells, that can be
induced to produce an embryo and then a complete plant,
has been demonstrated in many medicinal plant species
(Tripathi and Tripathi, 2003). Induction of callus In vitro
depends on concentration and type of PGRs added to the
basal medium (Ikeuchi et al., 2013). It also depends on
type of cultivars or explant and PGRs interaction. Saleh et
al. (2018) reported that the combination of 5 mg BA/L and
80 mg 2,4-D/L gave the highest percentage of callus
induction (88%) with a significant difference for the rest of
the treatment combinations that were used in their study
(Regardless of the effect of explants). But, Jasim et al.
(2009) reported that the medium supplemented with 50
mg/L of NAA and 3 mg/L of 2iP gave the highest
percentage of primary callus induced in date palm
cultivars.
Arumugam and Bhojwani (1990) reported the
development of somatic embryos from zygotic embryos of
Podophyllum hexandrum on MS medium containing BAP
and IAA. The efficient development and germination of
somatic embryos are prerequisites for commercial plantlet
production. Chand and Sahrawat (2002) reported the
somatic embryogenesis of Psoralea corylifolia L. from root
explants on medium supplemented with NAA (10.74 μM)
and BAP (2.2 μM). Rooting of shoots was best achieved
using different concentrations of auxins. For example, in
wood apple (Aegle marmelos L.), MS half-strength
medium supplemented with IAA (1mg/l) proved better
rooting (Yadav and Singh, 2011a). In Prosopis cineraria,
rooting was achieved on half strength MS medium
supplemented with 3.0 mg/l IBA (Kumar and Singh, 2009).
Acclimatization and Transfer of micro propagated
plantlets to the soil
Complete regenerated plantlets with sufficient roots were
gradually pulled out from the medium and immersed in
water to remove the remains of agar particles sticking to
the root system by using a fine brush and the plantlets
were transferred to pots containing a mixture of sterilized
soil and sand (3:1). The potted plantlets were covered with
a transparent polythene bag to ensure high humidity
around the plants. After about two weeks, the polythene
bags were removed for 3-4 hours daily to expose the
plants to the conditions of natural humidity for
acclimatization. These plants were shifted to bigger pots
after one month of its transfer and were maintained under
greenhouse conditions. Successful acclimatization and
field transfer of the in vitro regenerated plantlets have also
been reported in Peganum harmala (Goel et al., 2009),
Celastrus paniculatus (Lal and Singh, 2010). On the other
hand, Yadav and Singh (2011b) noted well-rooted
micropropagated plantlets of Albizia lebbeck L. were
acclimatized and successfully established in pots
containing sterilized soil and sand mixture (1:1) with 60%
survival rate under field conditions.
Ex Vitro field evaluation of acclimated plants
The process of transplantation and acclimatization of
micropropagated plants to soil environment is very
important for adaptation in medicinal plants.
Acclimatization of a micropropagated plant to a green
house or field environment is essential because
anatomical and physiological characteristics of in vitro
plantlets necessitate that they should be gradually
acclimatized to the field environment (Hazarika, 2003).
Successful acclimatization minimizes the percentage of
dead or damaged plants, enhancing the plant growth and
establishment. Dynamics of the process are related to the
acclimatized plant species and both in vitro and ex vitro
culture conditions (Pospisilova et al., 1999). Now days,
mycorrhizal technology can be applied to reduce
transplantation shock during acclimatization, thus
increasing plant survival and establishment rates of
micropropagated medicinal plant species (Sharma et al.,
2008; Yadav et al., 2011).
Production of secondary metabolites from medicinal
plants
Plants produce several compounds that are not essential
for primary functions like growth, photosynthesis and
reproduction and are called secondary metabolites.
Secondary metabolites are used as pharmaceutical,
agrochemicals, aromatics and food additives (Rao and
Ravishankar, 2002). Plants derived compounds include
many terpenes, polyphenols, cardenolides, steroids,
alkaloids and glycosides (Matkowski, 2008). Tissue culture
offers an effective and potential alternative of metabolite
production because the number of secondary metabolites
produced in tissue cultures can be even higher than in
parent plants (Rao and Ravishankar, 2002).
Production of secondary metabolites in cell suspension
cultures has been reported from various medicinal plants.
For example, enhanced indole alkaloid biosynthesis in the
suspension culture of Catharanthus roseus has been
reported (Zhao et al., 2001). Pan et al. (2003) obtained
high yields of proteolytic enzymes from the callus tissue
culture of garlic (Allium sativum L.) on MS medium
supplemented with NAA and BAP (Pan et al., 2003).
Pradel et al (1997) observed that the biosynthesis of
cardenolides was maximal in the hairy root cultures of
Digitalis lanata compared to leaf. The production of
azadirachtin and nimbin is to be higher in cultured shoots
and roots of Azadirachta indica compared to field grown
plants (Srividya et al., 1998). Pande et al. (2002) reported
that the yield of lepidine from Lepidium sativum Linn
depends upon the source and type of explants.
SUMMARY AND CONCLUSIONS
Medicinal plants, since times immemorial, have been used
in virtually all cultures as a source of medicine. Herbal

5. Micropropagation of Medicinal Plants: Review
Int. J. Plant Breed. Crop Sci. 800
medicines serve the health needs of millions all over the
world, especially in developing countries. Medicinal plants
are also the source of modern medicine. Medicines in
common use, such as aspirin and digitalis, are derived
from plants. Currently trade in medicinal plants is growing
in volume and in exports. Global herbal market is growing
at a rate of seven percent per annum.
In vitro propagation or tissue culture of plants holds
tremendous potential for the production of high-quality
plant-based medicines. Protocols have been developed
for clonal multiplication of hundreds of plant species of
medicinal plants. The increased use of plant cell culture
systems in recent years is perhaps due to an improved
understanding of the secondary metabolite pathway in
economically important plants. Advances in plant cell
cultures could provide new means for the cost-effective,
commercial production of even rare or exotic plants, their
cells, and the chemicals that they will produce. In general,
there is good progress in protocol optimization of medicinal
plants in developed countries and India. However, there is
a limitation in developing countries like Ethiopia so; there
is a need for research in these areas to conserve and to
maximize the use of medicinal plants.
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Accepted 22 May 2020
Citation: Lemma DT, Banjaw DT, Megersa HG (2020).
Micropropagation of Medicinal Plants: Review.
International Journal of Plant Breeding and Crop Science,
7(2): 796-802.
Copyright: © 2020: Lemma et al. This is an open-access
article distributed under the terms of the Creative
Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium,
provided the original author and source are cited.