Fahad Al-Qurainy, Mohammad Nadeem ?, Salim Khan, Saleh Alansi, Mohamed Tarroum,Abdulhafed A.
Al-Ameri, Abdel-Rhman Z. Gaafar, Aref AlshameriDepartment of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabiaarticle infoArticle history:Received 12 March 2017Revised 27 June 2017Accepted 15 July 2017Available online xxxxKeywords:Reseda pentagynaEndemicTissue cultureConservationMicropropagationabstractReseda pentagyna is the only endemic species among the seven species of the genera Reseda found inSaudi Arabia. Probably no information is available on regeneration by conventional method of regenerationthrough seeds or cuttings. Therefore, alternative method of tissue culture was attempted to regenerateand multiply the plant. High shoot regeneration (14.44 shoots/explant) was obtained after fourweeks, when shoot cuttings cultured on MS containing BA at 1.0 mM.
Other cytokinins e.g., Kn, 2iP andTDZ found to be less effective in bud induction and shoot multiplication.
Individual shoots were rootedon MS medium supplemented with various auxins at 0.5–5.0 mM concentrations. The IBA (1.5 mM) supplementedMS media induced maximum (83.3%) rooting.
The plantlets were acclimatized and hardenedunder greenhouse conditions in plastic pots containing soil and farm yard manure with 95.0% success.The protocol developed would help to multiply the plant as well as conserve them in natural habitat.
This can also be utilized to obtain active constituents for pharmaceutics and genetic manipulations. 2017 The Authors. Production and hosting by Elsevier B.V.
on behalf of King Saud University. This is anopen access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. IntroductionSeven species of the genus Reseda are found in Saudi Arabia, viz.,R. pentagyna Abdallah & A.
G. Miller, R. arabica Boiss., R. lutea, R. albaR. aucheri Boiss.
, R. muricata C. Presl, and R. sphenocleoides Deflers(Chaudhary, 1999).
Among the seven species, R. pentagyna has anendemic status in Saudi Arabia and distributed widely in WadiSawawin, Northern Hijaz mountains and Tabuk of north westernparts of Saudi Arabia (Miller and Nyberg, 1994; Chaudhary,1999; Llewellyn et al., 2010). The presence of only 3–4 toothedcapsules in R. pentagyna differentiate it from R. stenostachya thatshow the occurrence of 5–6 toothed capsules.
The conservation of endemic, threatened and endangeredmedicinal species is vital for the future of humankind. A rich indiversity is found in the flora of Saudi Arabia, with numerous rareand endangered plants spreading to different genera of plant kingdom.There is continuous threat to the survival of these plantsbecause of the prevalent environmental conditions. Therefore,the number of threatened plant species are more and increasingyearly resulting from harsh conditions and anthropogenic activitiesKhan et al., 2012. Over-exploitation, loss of habitat, speedyurbanization, over-grazing, selective species extraction from wilds,and damage is leading to genetic erosion. Climatic conditions inArabian region is tough that has resulted in declining plant population,fragmented habitats, endangerment, narrowed geneticdiversity, rarity, poor regeneration and reproductive inefficiencies,are wide spread in the Kingdom of Saudi Arabia (Al-Farhan et al.
,2005).Hence, it is required to search for the development of alternativepropagation method for this important plant. To augment thisconventional as well as in vitro approaches can be implemented formass multiplication and value addition to the plant. In generalplants regenerates through seeds, a very few literature is availablehttp://dx.doi.org/10.1016/j.sjbs.
2017.07.0031319-562X/ 2017 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).? Corresponding author.E-mail addresses: [email protected] (F.
-R.Z. Gaafar),[email protected]
com (A. Alshameri).Peer review under responsibility of King Saud University.Production and hosting by ElsevierSaudi Journal of Biological Sciences xxx (2017) xxx–xxxContents lists available at ScienceDirectSaudi Journal of Biological Sciencesjournal homepage: www.
sciencedirect.comPlease cite this article in press as: Al-Qurainy, F., et al. Rapid plant regeneration, validation of genetic integrity by ISSR markers and conservation of Resedapentagyna an endemic plant growing in Saudi Arabia.
Saudi Journal of Biological Sciences (2017), http://dx.doi.org/10.1016/j.sjbs.
2017.07.0033.3. ISSR analysis of in vitro raised plantsMicropropagation is used to obtain uniform planting material.However, it is necessary to authenticate the clonal uniformity ofin vitro-raised plants to confirm the reliability of the protocol formass propagation. DNA based Molecular markers are more reproducibleas compared to the cytological, morphological and proteinmarkers as they have been long practiced for the identification ofvariations in tissue-cultured raised plantlets, genetic diversitystudy, plant identification and beyond.
Moreover, these markersare highly reproducible, heritable, reliable, detectible in all tissuesand easy to perform. The genetic fidelity was assessed among theclones of R. pentagyna and compared with their mother plant usingthe ISSR molecular markers. We used 15 primers for genetic fidelitytesting and out of these, 14 primers amplified the genomicfragments and gave reproducible bands. All primers produced themonomorphic bands among the all regenerated plantlets as resultsof two primers shown in (Figs. 2 and 3). Thus, regenerated plantletsof R.
pentagyna maintained the genetic stability even after subculturingof calli for long term duration on the same media. Ourresult was congruence in line with other researchers as they didnot find any genetic variations on tissue cultured raised plantletssuch as Dendrocalamus strictus (Roxb.) (Goyal et al., 2015), Bananacv Robusta (Chaudhary, 2015), Tylophora indica (Sharma et al.,2014) etc.
ISSR markers have been used for the assessment ofgenetic fidelity in the tissue cultured raised plants including Bambusabalcoa (Negi and Saxena, 2010). Simmondsia chinensis (Link)Schneider (Kumar et al., 2011); olive species (Brito et al., 2010);Capparis spinosa L. (Carra et al., 2012); etc. The application of ISSRmarker is very easy and it uses single primer in PCR reaction likeRAPD marker.
4. ConclusionThe present study describes an efficient protocol for directshoot regeneration of R. pentagyna. The genetic uniformity ofmicropropagated plants were analyzed by using ISSR confirms nogenetic variations in the plants regenerated through an in vitromultiplication system.
The in vitro-raised cultures could be usedas a source for obtaining bioactive compounds on a large scale.Thus the developed protocol for regeneration is a reliable and commercialmultiplication but also for conservation of elite clones of R.pentagyna and other genetic manipulation.AcknowledgmentsThe authors extend their appreciation to the Deanship of ScientificResearch at King Saud University for funding the work throughthe research group Project no. RGP-014.