Plants value such as R. chalepensis is making a

Plants are essential for the survival of human
as they considered the only source of energy beside their different uses as
food and flavors (Cowan, 1999). Moreover, there are about 50,000-80000 plant
species being used for medicinal purposes, these plants contain a lot of
medicinal compounds helping to cure a range of diseases from the common cold to
cancer and even HIV/AIDS (Singh, 2011). In developed countries, about 40% or
more of compounds used in pharmaceutical industries are directly or indirectly
derived from plants (Rout et al., 2000).

Ruta
chalepensis L. is a
perennial herb belongs to Rutaceae family, known by the common name fringed rue
and the Arabic name “Al-Shathap”. It is localized in the Mediterranean area
(Iauk et al., 2004) and widely distributed in the Kingdom of Saudi Arabia.
Nowadays, it is cultivated in many regions throughout the world especially in
temperate and equatorial countries (Günaydin and Savci, 2005). R.
chalepensis is a medicinal plant used in the traditional medicine for the
treatment of a variety of diseases, e.g. epilepsy, vertigo, colic, intestinal
worms, toxicity, headache and eye problems (Gonzalez-Trujano et al., 2006).

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Cultivation
of medicinal plants particularly with high medicinal value such as R.
chalepensis is making a new dimension in the field of agriculture (Supe et
al., 2011). The growing demand for medicinal herbs, habitat destruction for
agricultural purposes and low seed viability are major concerns for the
propagation of these plants. Encouraging cultivation of different medicinal
plants will significantly reduce the dependence on their wild existing
populations (Afolayan and Adebola, 2004). One of the possible methods of production
and multiplication of these medicinal plants is plant tissue culture techniques
(Krishnan et al., 2011; Pandey et al., 2013), which are useful for mass
multiplication of threatened medicinal plants (Rout et al. 2000; Krishnan et
al., 2011).

In vitro plant propagation (micropropagation) is the
most widely used application of tissue culture technology, and it has become an
important part of the commercial propagation of many plants. It can be induced
by several techniques include plant regeneration by organogenesis and/or somatic
embryogenesis (Davey and Anthony, 2010; Al-Mahdawe et al., 2013).

Artificial
seeds technology is a potential tool for an efficient and cost-effective
micropropagation system. Plant proliferation and conservation through the
production of artificial seeds has unraveled new sights in plant biotechnology
(Ahmad et al., 2012; Banu et al., 2014). They make a promising technique for
the propagation of transgenic plants, rare hybrids, non-seed producing plants
and polyploids with elite traits and for multiplying and conserving the elite
agricultural and endangered medicinal plant species, which are difficult to
regenerate through conventional methods and natural seeds (Capuano et al.,
1998; Saiprasad 2001).

Genetic
stability of in vitro regenerated plants is a critical aspect of plant
propagation which is imperative for their commercial utilization (Bhattacharyya
et al., 2014). Hence, the genetic stability of in vitro propagated
plants should be evaluated for developing proper programs for conservation of
true-to-type plants. Genetic stability can be assessed by studying chromosome
numbers, isozyme profile and polymerase chain reaction (PCR)-based molecular
markers like inter simple sequence repeats (ISSR), random amplified polymorphic
DNA (RAPD), simple sequence repeat (SSR), Sequence-related amplified
polymorphism (SRAP), restriction fragment length polymorphism (RFLP) and
amplified fragment length polymorphism (AFLP) (Devi et al., 2013; Rathore et
al., 2014; Butiuc-Keul et al., 2016). In combination with molecular markers
flow cytometry has been employed successfully to analyze the nuclear DNA
content, genome size and ploidy stability of in vitro regenerated plants
(Mallon et al., 2010; Ghimire et al., 2012; Vujovic et al., 2012; Faisal et
al., 2014).

Until
now, there are no evidence for the application of tissue culture in R. chalepensis.
Therefore, this study aims to establish and optimize of in vitro
regeneration and multiplication protocol of R. chalepensis, acclimatize
the regenerated plants to ex vitro condition, develop of synthetic seeds
for germplasm storage and exchange and to assess the genetic stability of
regenerated plants using difference molecular markers and flow cytometry.