Fahad the seven species of the genera Reseda

 Fahad Al-Qurainy, Mohammad Nadeem ?
, Salim Khan, Saleh Alansi, Mohamed Tarroum,
Abdulhafed A. Al-Ameri, Abdel-Rhman Z. Gaafar, Aref Alshameri
Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
article info
Article history:
Received 12 March 2017
Revised 27 June 2017
Accepted 15 July 2017
Available online xxxx
Keywords:
Reseda pentagyna
Endemic
Tissue culture
Conservation
Micropropagation
abstract
Reseda pentagyna is the only endemic species among the seven species of the genera Reseda found in
Saudi Arabia. Probably no information is available on regeneration by conventional method of regeneration
through seeds or cuttings. Therefore, alternative method of tissue culture was attempted to regenerate
and multiply the plant. High shoot regeneration (14.44 shoots/explant) was obtained after four
weeks, when shoot cuttings cultured on MS containing BA at 1.0 mM. Other cytokinins e.g., Kn, 2iP and
TDZ found to be less effective in bud induction and shoot multiplication. Individual shoots were rooted
on MS medium supplemented with various auxins at 0.5–5.0 mM concentrations. The IBA (1.5 mM) supplemented
MS media induced maximum (83.3%) rooting. The plantlets were acclimatized and hardened
under 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 an
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
Seven species of the genus Reseda are found in Saudi Arabia, viz.,
R. pentagyna Abdallah & A.G. Miller, R. arabica Boiss., R. lutea, R. alba
R. aucheri Boiss., R. muricata C. Presl, and R. sphenocleoides Deflers
(Chaudhary, 1999). Among the seven species, R. pentagyna has an
endemic status in Saudi Arabia and distributed widely in Wadi
Sawawin, Northern Hijaz mountains and Tabuk of north western
parts of Saudi Arabia (Miller and Nyberg, 1994; Chaudhary,
1999; Llewellyn et al., 2010). The presence of only 3–4 toothed
capsules in R. pentagyna differentiate it from R. stenostachya that
show the occurrence of 5–6 toothed capsules.
The conservation of endemic, threatened and endangered
medicinal species is vital for the future of humankind. A rich in
diversity is found in the flora of Saudi Arabia, with numerous rare
and endangered plants spreading to different genera of plant kingdom.
There is continuous threat to the survival of these plants
because of the prevalent environmental conditions. Therefore,
the number of threatened plant species are more and increasing
yearly resulting from harsh conditions and anthropogenic activities
Khan et al., 2012. Over-exploitation, loss of habitat, speedy
urbanization, over-grazing, selective species extraction from wilds,
and damage is leading to genetic erosion. Climatic conditions in
Arabian region is tough that has resulted in declining plant population,
fragmented habitats, endangerment, narrowed genetic
diversity, 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 alternative
propagation method for this important plant. To augment this
conventional as well as in vitro approaches can be implemented for
mass multiplication and value addition to the plant. In general
plants regenerates through seeds, a very few literature is available
http://dx.doi.org/10.1016/j.sjbs.2017.07.003
1319-562X/ 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. Al-Qurainy), [email protected]
(M. Nadeem), [email protected] (S. Khan), [email protected]
(S. Alansi), [email protected] (M. Tarroum),
[email protected] (A.A. Al-Ameri), [email protected] (A.-R.Z. Gaafar),
[email protected] (A. Alshameri).
Peer review under responsibility of King Saud University.
Production and hosting by Elsevier
Saudi Journal of Biological Sciences xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Saudi Journal of Biological Sciences
journal homepage: www.sciencedirect.com
Please cite this article in press as: Al-Qurainy, F., et al. Rapid plant regeneration, validation of genetic integrity by ISSR markers and conservation of Reseda
pentagyna an endemic plant growing in Saudi Arabia. Saudi Journal of Biological Sciences (2017), http://dx.doi.org/10.1016/j.sjbs.2017.07.003

3.3. ISSR analysis of in vitro raised plants
Micropropagation is used to obtain uniform planting material.
However, it is necessary to authenticate the clonal uniformity of
in vitro-raised plants to confirm the reliability of the protocol for
mass propagation. DNA based Molecular markers are more reproducible
as compared to the cytological, morphological and protein
markers as they have been long practiced for the identification of
variations in tissue-cultured raised plantlets, genetic diversity
study, plant identification and beyond. Moreover, these markers
are highly reproducible, heritable, reliable, detectible in all tissues
and easy to perform. The genetic fidelity was assessed among the
clones of R. pentagyna and compared with their mother plant using
the ISSR molecular markers. We used 15 primers for genetic fidelity
testing and out of these, 14 primers amplified the genomic
fragments and gave reproducible bands. All primers produced the
monomorphic bands among the all regenerated plantlets as results
of two primers shown in (Figs. 2 and 3). Thus, regenerated plantlets
of R. pentagyna maintained the genetic stability even after subculturing
of calli for long term duration on the same media. Our
result was congruence in line with other researchers as they did
not find any genetic variations on tissue cultured raised plantlets
such as Dendrocalamus strictus (Roxb.) (Goyal et al., 2015), Banana
cv Robusta (Chaudhary, 2015), Tylophora indica (Sharma et al.,
2014) etc. ISSR markers have been used for the assessment of
genetic fidelity in the tissue cultured raised plants including Bambusa
balcoa (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 ISSR
marker is very easy and it uses single primer in PCR reaction like
RAPD marker.
4. Conclusion
The present study describes an efficient protocol for direct
shoot regeneration of R. pentagyna. The genetic uniformity of
micropropagated plants were analyzed by using ISSR confirms no
genetic variations in the plants regenerated through an in vitro
multiplication system. The in vitro-raised cultures could be used
as a source for obtaining bioactive compounds on a large scale.
Thus the developed protocol for regeneration is a reliable and commercial
multiplication but also for conservation of elite clones of R.
pentagyna and other genetic manipulation.
Acknowledgments
The authors extend their appreciation to the Deanship of Scientific
Research at King Saud University for funding the work through
the research group Project no. RGP-014.

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