Plants value such as R. chalepensis is making a

Plants are essential for the survival of humanas they considered the only source of energy beside their different uses asfood and flavors (Cowan, 1999). Moreover, there are about 50,000-80000 plantspecies being used for medicinal purposes, these plants contain a lot ofmedicinal compounds helping to cure a range of diseases from the common cold tocancer and even HIV/AIDS (Singh, 2011). In developed countries, about 40% ormore of compounds used in pharmaceutical industries are directly or indirectlyderived from plants (Rout et al., 2000).Rutachalepensis L.

is aperennial herb belongs to Rutaceae family, known by the common name fringed rueand 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 intemperate and equatorial countries (Günaydin and Savci, 2005). R.chalepensis is a medicinal plant used in the traditional medicine for thetreatment of a variety of diseases, e.g. epilepsy, vertigo, colic, intestinalworms, toxicity, headache and eye problems (Gonzalez-Trujano et al.

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, 2006).Cultivationof medicinal plants particularly with high medicinal value such as R.chalepensis is making a new dimension in the field of agriculture (Supe etal., 2011). The growing demand for medicinal herbs, habitat destruction foragricultural purposes and low seed viability are major concerns for thepropagation of these plants.

Encouraging cultivation of different medicinalplants will significantly reduce the dependence on their wild existingpopulations (Afolayan and Adebola, 2004). One of the possible methods of productionand multiplication of these medicinal plants is plant tissue culture techniques(Krishnan et al., 2011; Pandey et al., 2013), which are useful for massmultiplication of threatened medicinal plants (Rout et al. 2000; Krishnan etal.

, 2011).In vitro plant propagation (micropropagation) is themost widely used application of tissue culture technology, and it has become animportant part of the commercial propagation of many plants. It can be inducedby several techniques include plant regeneration by organogenesis and/or somaticembryogenesis (Davey and Anthony, 2010; Al-Mahdawe et al., 2013).Artificialseeds technology is a potential tool for an efficient and cost-effectivemicropropagation system. Plant proliferation and conservation through theproduction of artificial seeds has unraveled new sights in plant biotechnology(Ahmad et al., 2012; Banu et al., 2014).

They make a promising technique forthe propagation of transgenic plants, rare hybrids, non-seed producing plantsand polyploids with elite traits and for multiplying and conserving the eliteagricultural and endangered medicinal plant species, which are difficult toregenerate through conventional methods and natural seeds (Capuano et al.,1998; Saiprasad 2001).Geneticstability of in vitro regenerated plants is a critical aspect of plantpropagation which is imperative for their commercial utilization (Bhattacharyyaet al., 2014). Hence, the genetic stability of in vitro propagatedplants should be evaluated for developing proper programs for conservation oftrue-to-type plants. Genetic stability can be assessed by studying chromosomenumbers, isozyme profile and polymerase chain reaction (PCR)-based molecularmarkers like inter simple sequence repeats (ISSR), random amplified polymorphicDNA (RAPD), simple sequence repeat (SSR), Sequence-related amplifiedpolymorphism (SRAP), restriction fragment length polymorphism (RFLP) andamplified fragment length polymorphism (AFLP) (Devi et al., 2013; Rathore etal., 2014; Butiuc-Keul et al.

, 2016). In combination with molecular markersflow cytometry has been employed successfully to analyze the nuclear DNAcontent, genome size and ploidy stability of in vitro regenerated plants(Mallon et al., 2010; Ghimire et al., 2012; Vujovic et al., 2012; Faisal etal.

, 2014).Untilnow, there are no evidence for the application of tissue culture in R. chalepensis.Therefore, this study aims to establish and optimize of in vitroregeneration and multiplication protocol of R.

chalepensis, acclimatizethe regenerated plants to ex vitro condition, develop of synthetic seedsfor germplasm storage and exchange and to assess the genetic stability ofregenerated plants using difference molecular markers and flow cytometry.