Skip to main content
NCBI home page
Saudi Journal of Biological Sciences logoLink to Saudi Journal of Biological Sciences
. 2011 Nov 28;19(1):1–11. doi: 10.1016/j.sjbs.2011.11.001

Biotechnological approaches for conservation and improvement of rare and endangered plants of Saudi Arabia

Salim Khan 1,⁎, Fahad Al-Qurainy 1, Mohammad Nadeem 1
PMCID: PMC3730899  PMID: 23961155

Abstract

Genetic variation is believed to be a prerequisite for the short-and long-term survival of the plant species in their natural habitat. It depends on many environmental factors which determine the number of alleles on various loci in the genome. Therefore, it is important to understand the genetic composition and structure of the rare and endangered plant species from their natural habitat to develop successful management strategies for their conservation. However, rare and endangered plant species have low genetic diversity due to which their survival rate is decreasing in the wilds. The evaluation of genetic diversity of such species is very important for their conservation and gene manipulation. However, plant species can be conserved by in situ and in vitro methods and each has advantages and disadvantages. DNA banking can be considered as a means of complimentary method for the conservation of plant species by preserving their genomic DNA at low temperatures. Such approach of preservation of biological information provides opportunity for researchers to search novel genes and its products. Therefore, in this review we are describing some potential biotechnological approaches for the conservation and further manipulation of these rare and endangered plant species to enhance their yield and quality traits.

Keywords: Conservation, DNA bank, Genetic diversity, Molecular marker

1. Introduction

The Arabian Peninsula is one of the richest biodiversity area which comprises medicinal herbs, shrubs and trees. The medicinal plants of the Kingdom have potential diversity (Rahman et al., 2004), their usage to cure numerous human ailments is ancient and still available among the tribal, local people and traditional healers. The four fifths of the Arabian Peninsula are found in 225,000 km−2 area of Saudi Arabia which is a rich area of medicinal herbs, and the use of these in folk medicine has existed since time immemorial.

The flora of Saudi Arabia is suffering from various threats such as inconsistent weather, altered soil pH and composition, heavy metals stress, drought, salinity, extreme temperatures, altitudes and increased habitat fragmentation. Due to these reasons, their conservation and further improvement through biotechnological approaches are unavoidable. Their conservation became part of the governmental policy as early as in 1970s, and the Kingdom of Saudi Arabia joined the Convention on Biological diversity (CBD) in 2001. The CBD highlights three objectives: conservation of biological diversity, sustainable use of its components, and equitable sharing of benefits arising from the use of genetic resources. There are some plant genetic resources (PGR) in the Kingdom of Saudi Arabia in which the terrestrial ecosystems have small numbers of species with little inbuilt redundancy. The government of Saudi Arabia has introduced a number of policies for promoting natural resource management aimed at protecting the environment and its biodiversity and using the natural resources in a framework that allows the benefits to be shared among all stakeholders. However, the flora of Saudi Arabia is the admixture of the elements of Asia, Africa and Mediterranean region. The Southwestern and most of the Western coastal plains of this country fall under the Somalia-Masai phytogeographical region (Chaudhury and Al-Jowaid, 1999) and some places such as Asir, Hijaz and the Western mountainous area have more diversity which may be due to greater rainfall and altitude (Collenette, 1998). The natural habitat of Saudi Arabia has endemic (242) plants, rare and endangered (600) out of 2250 which need conservation and sustainable development (Collenette, 1998; NCWCD, 1998; Al-Farhan, 2000). Diversity in vegetation is generated due to the impact of various physiographic factors coupled with diverse climatic variations. The few species survive successfully in ex situ conservation; in situ conservation is difficult for most of the plant species due to habitat fragmentation, invasion of alien species as well as changes in climate. However, environmental factors are responsible for genetic diversity in a natural habitat therefore; in situ conservation is the best option for their conservation. There are many advantages of such conservation including genetic exchange through random mating and recombination, accumulation of beneficial mutation and transfer among the populations, competition among populations which helps for their better adaptation and greater genetic diversity (Engles, 2001; Vijayan et al., 2011). Various strategies such as botanical gardens, GenBank and seed banks should be implemented for the conservation of critically endangered species. The rare and endangered plants which may have potential genes should be conserved for future research in the Kingdom. As a prelude to conservation of various rare and endangered plant species from their natural habitat, identification of their genetic diversity and phylogenetic relationship using the molecular tools is indispensible.

2. Factors affecting genetic diversity

Study on genetic diversity is very important for rare and endangered plant species from their natural habitat. It will be helpful in formulating plans for management for preserving their genetic diversity and ensuring their long term survival. The natural habitat of a plant species determines its genetic diversity which is important for long-term survival and evolution under abiotic and biotic factors. Since, genetic diversity is influenced by numerous geographical factors due to which it constitutes subspecies, races or ecotypes in the nature (Sreekumar and Renuka, 2006). In addition, it is also affected by number of individuals and population size of a plant species. The species evolution in the natural habitat is affected by distribution of plant species. Genetic variation is necessary for short- and long-term survival of plant species in the wild conditions (Schonewald-Cox et al., 1983; Lande, 1988). The conservation of wild species is very important for their preservation of genetic diversity because their extinction rate is increasing directly and indirectly in response to anthropogenic activities and also by environmental factors.

Analysis of genetic structure of rare and endangered plant species is necessary which will help in their preservation either by ex situ or in situ. In situ conservation of plant species needs more attention to restore the suitable habitat based on the biological characteristics and ecological traits according to the natural habitat and to restore the efficient population size to minimize the loss of the genetic variation. To maintain the genetic diversity of a population, it is necessary to increase the population size by introduction of new populations in the place of those species which have been lost from the community. However, the natural habitat of a plant species is disturbed by numerous environmental factors which may lead to declination of the genetic diversity. Thus, loss in genetic diversity, inbreeding and extinction risk is resulted from the reduction in population size of a species (Frankham et al., 2002). The demographic history, geographic distribution range, mating systems, pollen and seed dispersal and successional stages are some of the factors that affect the genetic variation patterns among the plant populations (Hamrick and Godt, 1989; Nybom and Bartish, 2000; Nybom, 2004). Identification of genetic diversity within and among widespread, restricted and endangered congeneric species is necessary, prior to their conservation (Gitzendanner and Soltis, 2000) in vivo as well as in vitro. The sufficient genetic variability within and among populations of endangered species is brought through gene flow which is necessary for their long-term survival and evolution, and further, new selection pressure is resulted by environmental changes (Barrett and Kohn, 1991). The reduction in genetic diversity is resulted from the loss of alleles which may be due to the reduction in population sizes as genetic drift can induce a reduction in genetic variation and thus affects the survival of natural population (Reed and Frankham, 2003). The gene flow may occur among populations due to geographical proximity and ecological similarities, however, low migration levels could cause a reduction in genetic variation and bring about some natural selection pressure. Single marker is not sufficient for the evaluation of genetic diversity however; other markers should be employed for accuracy of the results. Assessment of genetic diversity would be more accurate if morphological, biochemical, phytochemical and DNA based markers are employed in the study.

3. Biotechnological approaches

A number of biotechnological approaches are being used for the conservation and improvement of plant species for desired traits and each of them has some advantages and disadvantages. Some important techniques which have great potential in the determination of genetic diversity have been used in many plant species including rare and endangered species.

3.1. Molecular marker technology

3.1.1. Biochemical marker

Different variants of the same enzymes having identical or similar functions are known as isozymes (or isoenzymes). They are powerful tools for the study of genetic variability within and between populations of plants and animals. Isozymes have been used in taxonomy, genetic, evolutionary and ecological studies, identification of cultivars and lines (Peirce and Brewbaker, 1973; Mondini et al., 2009). They are especially useful when several taxa, accessions and individuals are to be compared, as the assumption of homology is more accurate than with some DNA markers. Isozyme electrophoresis has been successfully used to identify clones and to examine the clonal structure of plant populations (Johanson et al., 1996; Lehmann, 1997).

3.1.2. Phytochemical markers

The discovery of novel compounds (phytochemicals) from wild plant species is an achievement toward the enhancement of the eradication of the human diseases. With the advancement of modern techniques such as mass spectrometry (MS) and nuclear magnetic resonance spectrometry (NMR) combined with separation techniques facilitated the identification and structural elucidation of molecules. These phytochemical analyses are valuable tools for taxonomic differentiation within species or for evaluating the effect of environmental factors (Hawkes, 1992). Metabolomics has been used to distinguish individual species within a genus (Heinrich, 2008; Tianniam et al., 2010), and also to discriminate the regional origins of particular species (Kang et al., 2008). Variation in biosynthesis of these metabolites could be a result from both genetic and environmental factors, which play important roles in the development of phenotypic variations in plants.

3.1.3. DNA based markers

DNA based markers either PCR based or non-PCR based provide genetic structure of the plant species and have been used in various fields such as embryology, genetic engineering, physiology, taxonomy etc. These markers are helpful in the identification of plant species and their cultivars to clarify errors occurred with the herbs in the local markets (Kiran et al., 2010). However, molecular linkage maps are being used successfully in many crop species for their improvement by locating the potential genes which control important traits in the plants. Some DNA markers assessed genetic diversity based on polymorphism. The polymorphism may be defined as simultaneous occurrence of multiple phenotypic forms of a trait attributable to the alleles of a single gene or the homolog of a single chromosome within or between populations. Various loci are amplified with the use of various markers and help in the evaluation of genetic diversity about the concerned plant species. The potential markers which are being used in the evaluation of genetic diversity for a plant genome are given below.

3.1.3.1. Random amplified polymorphic DNA (RAPD)

RAPDs are very quick and easy to develop due to the arbitrary sequence of the primers. It resolves most of the technical obstacle owing to its cost effective and easy to perform (Welsh and McClelland, 1990; Williams et al., 1990). Therefore, RAPDs have been extensively used in assessing genetic relationship among various accessions of different plant species (Khan et al., 2009a,b, 2010, 2011a,b).

3.1.3.2. Sequence characterized amplified region (SCAR)

The RAPD marker(s) can be converted into sequence characterized amplified regions (SCAR) (Paran and Michelmore, 1993), which is monolocus and more specific. These markers are reproducible and less sensitive to reaction conditions for amplification. These markers are based on sequencing of the unique band. The unique band is selected for the designing of longer primers that specifically binds to same loci of exact molecular weight as amplified in the PCR (Kiran et al., 2010; Al-Qurainy et al., 2011b).

3.1.3.3. Simple sequence repeat (SSR)

These are tandemly repeated DNA sequences that occur throughout the eukaryotic genome. The length variation in this marker is due to the DNA polymerase slippage during the replication of the SSR region (Levinson and Gutman, 1987). The frequency of this marker in the plant genome is estimated as one in every 6–7 kb (Cardle et al., 2000) thus, these markers show a high level of allelic diversity. The allelic polymorphism and co-dominant nature revealed by SSR markers have provided detailed information on genetic structure and gene flow in natural plant populations. The genomic library or SSR enriched library is important in searching the SSRs in eukaryotic genomes for which public DNA sequence data is lacking. SSR marker has been used for the assessment of genetic diversity in a numerous crops such as Psathyrostachys huashanica, Zea maize, Apium graveolens, Prunus domestica L., cherry plum (P. cerasifera Ehrh.) and sloe (P. spinosa L.) (Liu et al., 2010, 2010a; Wang et al., 2011; Horvath et al., 2011). Despite the advantages of these markers, their development is time consuming for a plant species for which little DNA sequence information is available in the public database.

3.1.3.4. Inter simple sequence repeat (ISSR)

ISSR marker is used for the evaluation of genetic diversity in the plant species where the sequence information is limited (Zietkiewicz et al., 1994; Al-Qurainy, 2010). The nucleotide repeats (inter simple sequence repeats) are distributed throughout the genome and has potential discriminatory power which has been seen in the clonal plant species for the assessment of genetic diversity (Camacho and Liston, 2001; Wang et al., 2004). The different plant species and their populations such as Monimopetalum chinens, Gynostemma pentaphyllum and Heptacodium miconioides which are endangered species of China were assessed by ISSR marker (Xie et al., 2005; Wang et al., 2008; Jin and Li, 2007). The critically endangered tree Dimorphandra wilsoni and widely distributed species D. mollis have been used for the identification of genetic diversity and structure for their conservation (Viana e Souza and Lovato, 2010; Vicente et al., 2011). ISSRs markers are quick, easy to handle and more reproducible than RAPDs markers (Wu et al., 2010).

3.1.3.5. Restriction fragment length polymorphism (RFLP)

The principle of RFLP is based on the variation in the restriction site of the enzyme which produces different restriction fragments on digestion. Being a co-dominant marker, it can detect coupling phase of DNA molecules as DNA fragments from all homologous chromosomes are detected. Due to highly specific, however, it is quite tedious and expensive since it requires large amount of purified DNA and an expertise of handling the radioactive substances. Genetic and chemical diversity of Eleutherococcus senticosus has been assessed by PCR-RFLP based on chloroplast trnK intron sequence (Zhu et al., 2011).

3.1.3.6. Amplified fragment length polymorphism (AFLP)

This technique is particularly useful for evaluating genetic diversity in those plant species where prior genetic information is not available. It is the modified form of the RFLP and uses PCR for the amplification of the restriction fragments generated on digestion of genomic DNA with restriction endonucleases. More variation in the restriction site, more polymorphism would be generated. However, genetic diversity is very important for the survival of the plant species in their natural habitat which is evaluated by AFLP marker in many plant species such as Hibiscus rosa-sinensis (Braglia et al., 2010), Ocimum spp. (Moghaddam et al., 2011), Punica granatum L. (Moslemi et al., 2010), Thai bananas (Wongniam et al., 2010), Aegiceras corniculatum (Deng et al., 2009).

3.1.3.7. Selective amplification of microsatellite polymorphic loci (SAMPL)

It is the modified form of the AFLP technique and has been used for the analysis of highly variable microsatellite regions of eukaryotic genomes. SAMPL has been reported to be more powerful than AFLPs in discriminating between closely related individuals in several plant complexes (Singh et al., 2002), assessing genetic diversity in Terminalia arjuna (Sarwat et al., 2011), Garnacha Grapevine (Meneghetti et al., 2011) and Origanum vulgare (Azizi et al., 2009). In another study, SAMPL was found to be more efficient than AFLP in differentiating closely related accessions of Azadiracta indica (Singh et al., 2002).

3.1.3.8. Single nucleotide polymorphism (SNP)

Single nucleotide polymorphisms (SNPs) are the most abundant sequence variations in the plant genome which are widely used as molecular genetic markers in cultivar identification, genetic diversity studies and linkage mapping, characterization and phylogenetic studies, marker-assisted breeding, positional cloning and genetic comparative mapping (Dong et al., 2010; Troggio et al., 2007). These markers occur in unlimited numbers, and such differences in nucleotide sequences between individuals in single copy DNA are potentially useful markers. Genetic diversity was assessed in wheat accessions using SNPs marker based on stress responsive gene TaSnRK2.7-B (Zhang et al., 2011).

3.1.3.9. Expressed sequence tagged (EST)

ESTs are used for full genome sequencing and mapping programs underway a number of organisms for identifying active genes thus helping in identification of diagnostic markers. Moreover, an EST that appears to be unique assists to isolate new genes of beneficial agronomic traits. Once generated, they are useful in cloning specific genes of interest and synteny mapping of functional genes in various related organisms. EST-SSR marker has been used for the assessment of genetic diversity in Chinese orchid cultivars (Huang et al., 2010), Chinese wild almond and Amygdalus nana (Tahan et al., 2009), Soja (Liu et al., 2010b), Listada de Gandía (Munoz-Falcon et al., 2011) and Dactylis glomerata L. (Xie et al., 2010).

3.1.3.10. Single strand conformation polymorphism (SSCP)

This technique is used for the identification of point mutation and also for polymorphism. The heterozygosity is easily detected by this technique as the mobility of the single stranded DNA changes with change in its GC content due to its conformational changes. In plants, however, it is not well developed although its application can be exploited in discriminating progenies, once suitable primers are designed for agronomically important traits.

3.1.3.11. Cleaved amplified polymorphic sequence (CAPS)

CAPS markers are based on STSs derived from ESTs marker and also known as PCR-RFLP. Unlike RAPD markers, the CAPS marker is a PCR based co-dominant marker which is reproducible and easier to manipulate in marker assisted selection (MAS) (Caranta et al., 1999). Generally, CAPS marker is a PCR-based marker in which a restriction site is present in only one of the two amplified sequences and this difference can be due to a SNP marker (Nemri et al., 2007). Thus, CAPS markers are required for genotyping, positional or map based cloning and molecular identification studies (Spaniolas et al., 2006). The CAPS marker has been successfully used for the cultivar identification of strawberry, which is a vegetatively propagated polyploid plant like the sweet potato (Kunihisa et al., 2003, 2005). Similarly, citrus and sweet potato cultivars have been identified by the CAPS marker (Amar et al., 2011; Tanaka et al., 2010).

3.2. Additional markers

3.2.1. Internal transcribed spacer (ITS) sequences

ITS markers are used for the identification of plant species based on the sequence variation. These sequences are varied from species to species in the plant Kingdom and have different rate of evolution, therefore they become favored markers in evolutionary studies in different taxonomic levels (Gulbitti-Onarici et al., 2009). The nrDNA region has frequent insertions/deletions which can be phylogenetically informative (Baldwin et al., 1995). Internal transcribed spacer sequences (ITS) of nrDNA have been widely used for resolving phylogenetic relationships in many plant species (Gulbitti-Onarici et al., 2009; Choo et al., 2009; Al-Qurainy et al., 2011), molecular authentication of herbal materials (Zhang et al., 2007), genetic diversity assessment (Mondini et al., 2009), intra-specific variation study (Haque et al., 2009), and DNA barcoding (Zuo et al., 2010).

3.2.2. Chloroplast spacer sequences

The chloroplast (cp) genome of most angiosperm is circular and has size from 120 to 160 kb in length. A total of 132 cp genome has been sequenced up to now and out of them 22 are from monocot, and date palm. Khalas cultivar is the first member of Arecaceae family which has been sequenced (Jansen et al., 2005; Yang et al., 2010).The sequencing based DNA barcode generated from nuclear and/or chloroplast DNA has many advantages over polymorphic markers. The barcoding approach also has great potential for identifying plants and their adulterants easily (Jaakola et al., 2010; Yao et al., 2009; Al-Qurainy et al., 2011a). The genetic differentiation in the populations of Notopterigium forbesii was examined using chloroplast trnH-psbA intergenic spacer sequences (Zhou et al., 2010) and found low level of differentiation due to abundance of ancestral haplotype sharing and the high number of private haplotype.

3.2.3. DNA microarray

To date, the main applications of microarrays are to monitoring simultaneous gene expression, DNA variation analyses for the identification and genotyping of mutations. DNA microarray uses gene chips or microarrays to examine a multitude of loci at one time. To decrease complexity, cDNA or RNA is hybridized. The DNA on slide is probed with a fluorescently labeled probe and then scanned for polymorphisms. This technique is robust and high-throughput while eliminating the need for either agarose or polyacrylamide gels.

3.3. Plant DNA bank

Plant DNA bank has emerged as new resources with great potential for characterization and utilization of biodiversity worldwide. It can be used for plant breeding, biotechnology and biodiversity evaluation (Hodkinson et al., 2007). In the Kingdom of Saudi Arabia, plant biodiversity of major and minor crops is under serious threat from various factors such as high- and low-temperature, altitudes, salinity, habitat fragmentation, exposure to pollution, drought etc. The DNA collections have become important resources in worldwide and effort to address the biodiversity crisis, manage the world’s genetic resources and maximize their potential. However, analysis of DNA sequence is a powerful method by which the identification and delimitation of species, higher taxa can be done via DNA taxonomy and DNA barcoding. There is an urgent need in Saudi Arabia for more formal co-operation and communication between existing research institutes in the world, so that experiences and expertise can be shared. Such DNA bank in the Kingdom of Saudi Arabia would be of many advantages for the plant molecular biology researchers working on phylogeny, gene identification and its manipulation for agronomic traits. The database is indispensable for the DNA bank which is pivotal for the efficient utilization of the germplasm. Such DNA database is available from fully developed DNA bank worldwide and now Kingdom of Saudi Arabia is aware to establish plant DNA bank for conservation of vast diversity of flora. The databases concerned to DNA bank have numerous information regarding plant family, genus, author of DNA extraction, and type of DNA product or marker generated from the DNA which will be available via the Worldwide-Web for easy access. The recipient may utilize the material for breeding, research and training but cannot claim ownership over the material, or exchange it with others, without a legally binding written agreement. The establishment of DNA bank in the Kingdom of Saudi Arabia will facilitate the screening of plant by making DNA from large numbers of plant species available widely. To access and benefit sharing nationally and internationally, DNA bank requires the development of appropriate policies. Members of the organization on DNA banking network often meet to share technical knowledge among themselves on cryo storage of DNA and DNA-rich materials (Adams and Adams, 1992, 1998). Once DNA bank is being developed in the Kingdom of Saudi Arabia, we will able to construct genomic library, cloning of novel genes and molecular markers besides intact DNA. Due to inconsistent environmental conditions, therefore, there is an immediate requirement to establish a plant DNA bank in the Kingdom of Saudi Arabia to speed up the research in the field of plant science.

3.4. Unconventional biotechnological methods

3.4.1. Micropropagation

Plant micro-propagation is a biotechnological approach for the conservation of plant species and even for recalcitrant seeds of economically important crops. It may help in the production of virus free plants and is successful for those plant species which have difficulties in propagation using conventional methods. It is an in situ conservation of plant species having special phenotypic characters by introduction of regenerated plantlets directly into its natural habitat (Juliani et al., 2010). Nowadays, the trend in plant conservation is to combine ex situ and in situ species within an integrated conservation program including the collaboration among scientific or plant breeding organizations (Akeroyd, 2006). In a short period of time and limited space, it allows the production of numerous plant species. It is exploited for the production of pathogen-free cloned plants for agriculture and forestry usages. From conservation point of view, the regenerated plantlets should have minimum somatic variations through micro-propagation method by reducing the number of sub-culturing and axillary bud or shoot tip culture. During in vitro culture, since cells receive sequential stimuli for proper growth and finally plantlets formation in a large number. However, in a gene pool of the cells there may be insertion, deletion or substitution which leads to the formation of somatic variations. The selection of the explants is very important for the successful micro-propagation which decides the final fate of the developed plantlets via callus or without formation of calluses. The micropropagation of Lavandula pedunculata was done for essential oil production (Zuzarte et al., 2010) without affecting natural resources. Generally, somaclonal variations are produced during calluses formation or from suspension cell culture which reduces the economic value of the regenerated plants (Borse et al., 2011). Somaclonal variation may be genetic (heritable) or epigenetic (due to gene silencing or gene inactivation) which may be produced from point mutation, gene duplication, changes in number and the structure of the chromosome and movement of transposable element (Jain, 2001).

3.4.2. Protoplast culture

It is a physical phenomenon and during fusion two or more protoplasts come in contact and adhere with one another either spontaneously or in the presence of fusion inducing agents. Some useful genes such as disease resistance, nitrogen fixation, rapid growth and more product formation rate, protein quality, frost hardiness, drought resistance, herbicide resistance, heat and cold resistances are possible to transfer from one species to another. Thus, such biotechnological approach has been used to combine genes from different organisms to create strains with desired properties. Para-sexual hybrid protoplasts can be obtained from two genetically different protoplasts isolated from the somatic cells and are experimentally fused to each other.

3.4.3. Somatic embryogenesis

The development of somatic embryo from a single or multi-cells is known as somatic embryogenesis. Plant genetic transformation and gene cloning depend on the plant tissue culture practices for their regeneration on suitable media. However, somatic embryos have been used as the starting material for all transformation methods and can be obtained from reproductive explants including ovaries, stigmas, anthers, and whole flowers (Kikkert et al., 2005; Morgana et al., 2004; Gambino et al., 2007; Prado et al., 2010). Plant propagation through somatic embryogenesis helps to obtain a large number of plants irrespective of seasons and provides several advantages over traditional methods. The developed plantlets through somatic embryogenesis (SE) are to be elite clones whereas zygotic embryo, are unable to produce elite clones. The main advantage of SE is the production of numerous plantlets from a single cell which provides an option for their screening and evaluation. However, the regeneration of plantlets depends on the type of explants, combination and concentrations of growth regulators and medium used at different stages of culture. Haploid plantlets can be developed through this method which has potential application in the mutation research. Earlier, plant breeders have many difficulties in the production of homozygous line through selfing but biotechnological tools have an important contribution in the production of homozygous line from gametic embryogenesis (Germana et al., 2011). The direct embryogenesis is the preferable choice for embryos development; because it avoids the dedifferentiated callus phase and thus produced plantlets are genetically stable.

3.4.4. Organogenesis

The de novo organ synthesis such as buds, roots and shoots from cultured tissues is called organogenesis. A high cytokinin to auxin ratio induces in vitro shoot organogenesis, while high auxin to cytokinin induces production of roots. Recently, new insights into cytokinin and auxin interactions during plant organogenesis have been reported, demonstrating a link between the distribution of these hormones and de novo organogenesis Benkova et al., 2009; Pernisova et al., 2009). Plant mature cells have the capability to reverse their state of differentiation and produce new organs under cultured conditions. In vitro organogenesis, dedifferentiation and redifferentiation phases are commonly characterized. The balance of exogenous auxin and cytokinin in the medium is essential for de novo organogenesis (Skoog and Miller, 1957). Three phases are characterized for the organogenesis; first explants respond to phytohormone, second quiescent cells re-enter in the cell cycle and cell fate is determined to form specific organ primordia, which is the key step during de novo organogenesis and in the final stage, the morphogenesis of in vitro organs. Thus, organogenesis entails the regulation of cell division, cell expansion, cell and tissue type differentiations, and patterning of the organ as a whole.

3.4.5. Micrografting

The rejuvenation of the mature scion shoot tip onto the juvenile root stock is called micro-grafting and it was studied in vivo as well as in vitro. This method was developed (Jonard, 1986) for the production of disease-free scions, the rejuvenation and/or the invigoration of mature shoot materials, the enhanced potential for true to type cloning mature plants, and the study of graft unions (Onay et al., 2003). The kinnow mandarian was regenerated virus free using in vitro micrografting (Singh et al., 2008). There is one limitation in the production of micrografted plantlets as browning of cut surfaces due to oxidation of phenolic compounds. However, this problem may be removed by dipping the shoot scion in the MS medium.

3.4.6. Cryopreservation

It is important for long-term preservation of clonally propagated plant species which produce recalcitrant seeds and cannot be readily conserved by conventional methods through seed preservation (Benson, 2004). Now it is applied to a diverse range of plant species and their tissues (Reed, 2008). These species are unable to produce true seeds but can propagate through grafting or cutting. Various cryopreservation methods are being used for various plant species such as vitrification, encapsulation–dehydration and encapsulation–vitrification (Kami et al., 2009). Before cryopreservation of plant material, dehydration is necessary to protect the cells from lethal intracellular freezing and, dehydration can occur during rapid cooling in liquid nitrogen (Popova et al., 2010; Chen et al., 2011). The cooling rate is also an important factor for successful cryopreservation because ultra freezing rate helps to avoid intracellular freezing. Sometimes genetic variations are generated in the cryopreserved material due to the free radical generation and it is presumed due to dehydration process (Sanchez et al., 2008; Martin et al., 2011). There are some limitations of this method for the preservation of such plant species. For instance, during the preservation of the plant tissues, methylation starts in the cell and it occurs in the plant mainly on cytosine (CpG and CpXpG) and forming 5-methyl-cytosine. Undoubtedly, genetic/physiological factors determine outcomes of the cryopreservation for which a better understanding for their predisposing actions would benefit cryopreservation protocol development and germplasm responses following recovery (Johnston et al., 2009). DNA methylation does not change the gene sequence and its function but its level of expression is changed. The genetic stability is maintained during cryopreservation and has been proved by molecular markers study (Liu et al., 2008a). The cryopreserved tissue may also be considered to be safer for exchange of germplasm between country and regions. It also helps in eradication of virus and phytoplasm from plant tissues, and making them free from pathogen (Feng et al., 2011).

3.5. Genetic manipulation for improvement of yield and quality traits

3.5.1. Agrobacterium tumefaciens-mediated transformation

Biotechnological tools are important for selecting, analyzing, multiplying and improving the plants (Khan et al., 2009a,b). Over the years, several techniques have been adopted for enhancing bioactive molecules in medicinal plants (Khan et al., 2009a,b). Combinatorial biosynthesis is a tool to combine genes from different organisms to produce bioactive compounds in plants. The basic concept of this approach is combining the metabolic pathways in different organisms on genetic level (Horinouchi, 2009). Agrobacterium rhizogenes possesses infecting plasmids called root-inducing plasmid or Ri plasmid with T-DNA. The plasmid infects plant cell and induces the production of root-like hairy structures called “hairy root disease”. The neoplastic (cancerous) roots produced upon infection have genetic and biochemical stability and a fast growth rate resulting in a large mass/medium ratio in a hormone free-media (Sivakumar, 2006; Srivastava and Srivastava, 2007). These genetically transformed root cultures can produce secondary metabolites in large amounts comparable with those in intact plants, and the transformed root lines can be a promising source for the constant and standardized production of secondary metabolites.

3.5.2. RNAi technology

The RNA interference (RNAi) technique can lower the enzyme activities, which can help to suppress the formation of unwanted natural compounds or channelize the metabolites into pathways leading to desired products. To date RNAi is a potential technique and is being used for the production of medicinal plants with potential traits for a particular marker compound which is needed for eradication of diseases. RNA interference (RNAi) technology has provided an alternative to block the activity of such enzymes that are not only encoded each by a multigene family but are also expressed across a number of tissues and developmental stages.

3.5.3. Molecular pharming for high production of bioactive compounds

Production of potential pharmaceutical compounds using plant transformation strategies and transient expression system such as agro-infilteration, virus infection, magnifection (Obembe et al., 2011) is known as molecular pharming or bio-pharming. Thus, it focuses mainly on the biosynthesis of proteins and secondary metabolites which are very useful for humans but they are costly in the market. Earlier, molecular pharming was used on transgenic animals for the production of high valued compounds which are being used at large scale for industrial purpose. Plants can act as factories for the biosynthesis of drugs or compounds which are very costly in the markets. The biosynthesis of compounds in plant based systems has many advantages over others. There are various bio-products such as vaccine, antigen, antibody, therapeutical and nutraceutical proteins, non-pharmaceutical plant derived proteins are being produced using molecular pharming (Obembe et al., 2011).

3.6. Conclusion and future prospects

Biotechnological tools are necessary for the conservation and improvement of the plant species mainly rare and endangered which have importance economically and medicinally. Genetic diversity is important for the survival of the plant species in their natural habitat. However, loss in genetic diversity of the plant may lead to decline its ability to cope with changing environment and demographic fluctuations both in short- and long-term. Once plant species are disappeared from their natural habitat, they cannot be regenerated again which is very difficult to reestablish their previous rich diversity. Therefore, these tools should be applied for the conservation and improvement of the various plant species in a number of ways to broaden the genetic diversity of the rare and endangered plants of Saudi Arabia. DNA banking is also a potential method for the conservation of biological information by preserving the genomic DNA at low temperatures and has been established in few countries. The DNA isolation is easy and can be used extensively for the characterization and utilization of biodiversity. The implementation of such biotechnological tools on rare and endangered plant species of Saud Arabia may help in revival of their previous genes and their products which have been disappeared or inactivated in natural habitat.

Acknowledgement

Authors are thankful to the National Plan for Science and Technology, (NPST) program, King Saud University, Riyadh, Saudi Arabia for providing fund (Project No.: 10-BIO1289-02).

References

  1. Adams R.P., Adams J.E. Academic; San Diego: 1992. Conservation of Plant Genes: DNA Banking and in vitro Biotechnology. [Google Scholar]
  2. Adams R.P., Adams J.E. Vol. 71. Springer; Berlin: 1998. (Conservation of plant genes III conservation and utilization of African plants, Monographs in Systematic Botany). [Google Scholar]
  3. Akeroyd J.R. Plant taxonomy and reintroduction. In: Leadlay E., Jury S., editors. Taxonomy and Plant Preservation. University Press; Cambridge, UK: 2006. pp. 221–227. [Google Scholar]
  4. Al-Farhan, A. H., 2000. An evaluation of the current status of the flora of Saudi Arabia. Country report presented at the 2nd Arabian plants subject group meeting, Abu Dhabi, May 2000.
  5. Al-Qurainy F. Application of inter simple sequence repeat (ISSR marker) to detect genotoxic effect of heavy metals on Eruca sativa (L) Afr. J. Biotechnol. 2010;9(4):467–474. [Google Scholar]
  6. Al-Qurainy F., Khan S., Ali M.A., Al-Hemaid F.M., Tarroum M., Ashraf M. Authentication of Ruta graveolens and its adulterant using internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA. Pak. J. Bot. 2011;43(3):1613–1620. [Google Scholar]
  7. Al-Qurainy F., Khan S., Al-Hemaid FM., Ali M.A., Tarroum M., Ashraf M. Assessing molecular signature for some potential date (Phoenix dactylifera L) cultivars from Saudi Arabia based on chloroplast DNA sequences rpoB and psbA-trnH. Int. J. Mol. Sci. 2011;12:6871–6880. doi: 10.3390/ijms12106871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Al-Qurainy F., Khan S., Tarroum M., Al-Hemaid F.M., Ali M.A. Molecular authentication of the medicinal herb Ruta graveolens (Rutaceae) and an adulterant using nuclear and chloroplast DNA markers. GMR. 2011;10(4):2806–2816. doi: 10.4238/2011.November.10.3. [DOI] [PubMed] [Google Scholar]
  9. Amar M.H., Biswas M.K., Zhang Z., Guo W.W. Exploitation of SSR, SRAP and CAPS-SNP markers for genetic diversity of citrus germplasm collection. Sci. Hortic. 2011;128:220–227. [Google Scholar]
  10. Azizi A., Wagner C., Honermeier B., Friedt W. Intra-specific diversity and relationship between subspecies of Origanum vulgare revealed by comparative AFLP and SAMPL marker analysis. Plant Syst. Evol. 2009;281:151–160. [Google Scholar]
  11. Baldwin B.G., Sanderson M.J., Porter J.M., Wojciechowski M.F., Campbell C.S., Donoghue M.J. The ITS region of nuclear ribosomal DNA: a valuable source of evidence on angiosperm phylogeny. Ann. Mol. Bot. Gard. 1995;82:247–277. [Google Scholar]
  12. Barrett S.C.H., Kohn J. The genetic and evolutionary consequences of small population size in plant: implications for conservation. In: Falk D., Holsinger K.E., editors. Genetics and Conservation of Rare Plants. Oxford University Press; 1991. pp. 3–30. [Google Scholar]
  13. Benkova E., Ivanchenko M.G., Friml J., Shishkova S., Dubrovsky J.G. A morphogenetic trigger: is there an emerging concept in plant developmental biology. Trends Plant Sci. 2009;14:189–193. doi: 10.1016/j.tplants.2009.01.006. [DOI] [PubMed] [Google Scholar]
  14. Benson E.E. Cryo-conserving algal and plant diversity: historical perspective and future challenges. In: Fuller B., Lane N., Benson E.E., editors. Life in the Frozen State. CRC Press; London, UK: 2004. pp. 299–328. [Google Scholar]
  15. Borse N.V., Chimote P., Jadhav A.S. Stability of micro-propagated Musa acuminata cv. Grand Naine over clonal generations: a molecular assessment. Sci. Hortic. 2011;129(3):390–395. [Google Scholar]
  16. Braglia L., Bruna S., Lanteri S., Mercuri A., Portis E. An AFLP-based assessment of the genetic diversity within Hibiscus rosa-sinensis and its place within the Hibiscus genus complex. Sci Hortic. 2010;123:372–378. [Google Scholar]
  17. Camacho F.J., Liston A. Population structure and genetic diversity of Botrychium pumicola (Ophioglossaceae) based on inter-simple sequence repeats (ISSR) Am. J. Bot. 2001;88:1065–1070. [PubMed] [Google Scholar]
  18. Caranta C., Thabuis A., Palloix A. Development of a CAPS marker for the Pvr 4 locus: a tool for pyramidine potyvirus resistance gene in pepper. Genome. 1999;42:1111–1116. [PubMed] [Google Scholar]
  19. Cardle L., Ramsay L., Milbourne D., Macaulay M., Marshall D., Waugh R. Computational and experimental characterization of physically clustered simple sequence repeats in plants. Genetics. 2000;156:847–854. doi: 10.1093/genetics/156.2.847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Chaudhury, S.A., Al-Jowaid A., 1999. Vegetation of the Kingdom of Saudi Arabia. Riyadh: Ministry of Agriculture and Water.
  21. Chen X.L., Li J.H., Xin X., Zhang Z.E., Xin P.P., Lu X.X. Cryopreservation of in vitro-grown apical meristems of Lilium by droplet-vitrification. South Afri. J. Bot. 2011;77:397–403. [Google Scholar]
  22. Choo B.K., Moon B.C., Ji Y., Kim B.B., Choi G., Yoon T., Kim H.K. Development of SCAR markers for the discrimination of three species of medicinal plants, Angelica decursiva (Peucedanum decursivum) Peucedanum praeruptorum and Anthricus sylvestris, based on the internal transcribed spacer (ITS) sequence and random amplified polymorphic DNA (RAPD) Biol. Pharm. Bull. 2009;32:24–30. doi: 10.1248/bpb.32.24. [DOI] [PubMed] [Google Scholar]
  23. Collenette S. Checklist of botanical species in Saudi Arabia. West Sussex (UK): Int. Asclepiad Soc. 1998:78. [Google Scholar]
  24. Deng S., Huang Y., He H., Tan F., Ni X., Jayatissa L.P., Hettiarachi S., Shi S. Genetic diversity of Aegiceras corniculatum (Myrsinaceae) revealed by amplified fragment length polymorphism (AFLP) Aquat. Bot. 2009;90:275–281. [Google Scholar]
  25. Dong J., Qing-liang Y., Fu-sheng W., Li C. The mining of citrus EST-SNP and its application in cultivar discrimination. Agric. Sci. China. 2010;9:179–190. [Google Scholar]
  26. Engles, J., 2001. Home gardens-a genetic resources perspective. In: Watson, J.W., Eyzaguirre, P.B. (Eds.), Home gardens and in situ conservation of plant genetic bresources in farming F systems. Proceedings of 2nd International Home Garden Workshop. Witzenhausen, Federal Republic of Germany, pp. 3–9.
  27. Feng C., Yin Z., Ma Y., Zhang Z., Chen L., Wang B., Li B., Huang Y., Wang Q. Cryopreservation of sweet potato (Ipomoea batatas) and its pathogen eradication by cryotherapy. Biotechnol. Adv. 2011;29:84–93. doi: 10.1016/j.biotechadv.2010.09.002. [DOI] [PubMed] [Google Scholar]
  28. Frankham R., Ballou J.D., Briscoe D.A. Cambridge University Press; 2002. Introduction to Conservation Genetics. [Google Scholar]
  29. Gambino G., Ruffa P., Vallania R., Gribaudo I. Somatic embryogenesis from whole flowers, anthers and ovaries of grapevine (Vitis spp.) Plant Cell Tissue Organ Cult. 2007;90:79–83. [Google Scholar]
  30. Germana M.A., Chiancone B., Padoan D., Barany I., Risueno M.C., Testillano P.S. First stages of microspore reprogramming to embryogenesis through anther culture in Prunus armeniaca L. Environ. Exp. Bot. 2011;71:152–157. [Google Scholar]
  31. Gitzendanner M.A., Soltis P.S. Patterns of genetic variation in rare and widespread plant congeners. Am. J. Bot. 2000;87:783–792. [PubMed] [Google Scholar]
  32. Gulbitti-Onarici S., Sancak C., Sumer S., Ozcan S. Phylogenetic relationships of some wild wheat species based on the internal transcribed spacer sequences of nrDNA. Curr. Sci. 2009;96:794–800. [Google Scholar]
  33. Hamrick J.L., Godt M.J.W. Allozyme diversity in plant species. In: Brown A.H.D., Clegg M.T., Kahler A.L., Weir B.S., editors. Plant Population Genetics. Sinauer; Sunderland: 1989. (Breeding and Genetic Resources). [Google Scholar]
  34. Haque I., Bandopadhyay R., Mukhopadhyay K. Intra-specific variation in Commiphora wightii populations based on internal transcribed spacer (ITS1-58S-ITS2) sequences of rDNA. Diversity. 2009;1:89–101. [Google Scholar]
  35. Hawkes J. In: The Potato Crop. 2nd ed. Harris P., editor. Chapman-Hall; London: 1992. pp. 13–64. [Google Scholar]
  36. Heinrich M. Ethnopharmacy and natural product research-multidisciplinary opportunities for research in the metabolomic age. Phytochem. Lett. 2008;1:1–5. [Google Scholar]
  37. Hodkinson T.R., Waldren S., Jan P., Kelleher C.T., Salamin K., Salamin N. DNA banking for plant breeding, biotechnology and biodiversity evaluation. J. Plant Res. 2007;120:17–29. doi: 10.1007/s10265-006-0059-7. [DOI] [PubMed] [Google Scholar]
  38. Horinouchi S. Combinatorial biosynthesis of plant medicinal polyketide by microorganisms. Curr. Opin. Chem. Biol. 2009;13(2):197–204. doi: 10.1016/j.cbpa.2009.02.004. [DOI] [PubMed] [Google Scholar]
  39. Horvath A., Balsemin E., Barbot J.C., Christmann H., Manzano G., Reynet P., Laigret F., Mariette S. Phenotypic variability and genetic structure in plum (Prunus domestica L.), cherry plum (P. cerasifera Ehrh.) and sloe (P. spinosa L.) Sci. Hort. 2011;129:283–293. [Google Scholar]
  40. Huang Y., Li F., Chen K. Analysis of diversity and relationships among Chinese orchid cultivars using EST-SSR markers. Biochem. Syst. Ecol. 2010;38:93–102. [Google Scholar]
  41. Jaakola L., Suokas M., Haggman H. Novel approaches based on DNA barcoding and high-resolution melting of amplicons for authenticity analyses of berry species. Food Chem. 2010;123:494–500. [Google Scholar]
  42. Jain S.M. Tissue culture derived variation in crop improvement. Euphytica. 2001;118:153–166. [Google Scholar]
  43. Jansen R.K., Raubeson L.A., Boore J.L., dePamphilis C.W., Chumley T.W. Methods for obtaining and analyzing whole chloroplast genome sequences. Methods Enzymol. 2005;395:348–384. doi: 10.1016/S0076-6879(05)95020-9. [DOI] [PubMed] [Google Scholar]
  44. Jin Z., Li J. Genetic differentiation in endangered Heptacodium miconioides Rehd. based on ISSR polymorphism and implications for its conservation. Forest Ecol. Manag. 2007;245:130–136. [Google Scholar]
  45. Johanson B.O., Jonsdottir I.S., Cronbcrg N. Clonal diversity and allozyme variation in populations of the arctic sedge Carex bigclowii (Cyperaceae) J. Ecol. 1996;84:449–459. [Google Scholar]
  46. Johnston J.W., Benson E.E., Harding K. Cryopreservation induces temporal DNA methylation epigenetic changes and differential transcriptional activity in Ribes germplasm. Plant Physiol. Biochem. 2009;47:123–131. doi: 10.1016/j.plaphy.2008.10.008. [DOI] [PubMed] [Google Scholar]
  47. Jonard R. Micrografting and its applications to tree improvement. In: Bajaj Y.P.S., editor. Biotechnology in Agriculture and Forestry. Trees I. Springer-Verlag; Berlin, Heidelberg, New York, Tokyo: 1986. pp. 31–48. [Google Scholar]
  48. Juliani H.R., Korochb A.R., Zygadloc J.A., Trippi V.S. Evaluation of micro-propagation for the introduction into cultivation and conservation of Lippia junelliana, an endemic aromatic plant from Argentina. Ind. Crop Prod. 2010;34:1353–1357. [Google Scholar]
  49. Kami D., Shi L., Sato T., Suzuki T., Oosawa K. Cryopreservation of shoot apices of hawthorn in vitro cultures originating from East Asia. Sci. Hort. 2009;120:84–88. [Google Scholar]
  50. Kang J., Lee S., Kang S., Kwon H.N., Park J.H., Kwon S.W., Park S. NMR-based metabolomics approach for the differentiation of ginseng (Panax ginseng) roots from different origins. Arch. Pharm. Res. 2008;31:330–336. doi: 10.1007/s12272-001-1160-2. [DOI] [PubMed] [Google Scholar]
  51. Khan M.Y., Aliabbas S., Kumar V., Rajkumar S. Recent advances in medicinal plant biotechnology. Indian J. Biotechnol. 2009;8:9–22. [Google Scholar]
  52. Khan S., Mirja K.J., Tayyab Md., Abdin M.Z. RAPD profile for authentication of medicinal plant Glycirrhiza glabra L. MAPSB. 2009;3(1):48–51. [Google Scholar]
  53. Khan S., Mirza K.J., Abdin M.Z. Development of RAPD markers for authentication of medicinal plant Cuscuta reflexa. Eur. Asia J. Bio. Sci. 2010;4(1):1–7. [Google Scholar]
  54. Khan S., Mirza K.J., Al-Qurainy F., Abdin M.Z. Authentication of the medicinal plant Senna angustifolia by RAPD profiling. SJBS. 2011;18:287–297. doi: 10.1016/j.sjbs.2011.03.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Khan S., Mirza K.J., Tyagi M.R., Abdin M.Z. Development of RAPD markers for authentication of Ruta graveolens (L) and its adulterant. MAPSB. 2011;5(1):58–61. [Google Scholar]
  56. Kikkert J.R., Striem M.J., Vidal J.R., Wallace P.G., Barnard J., Reisch B.I. Long term study of somatic embryogenesis from anthers and ovaries of 12 grapevine (Vitis sp.) genotypes in vitro cell. Dev. Biol. Plant. 2005;41:232–239. [Google Scholar]
  57. Kiran U., Khan S., Mirza K.J., Ram M., Abdin M.Z. SCAR markers: a potential tool for authentication of herbal drugs. Fitoterapia. 2010;81:969–976. doi: 10.1016/j.fitote.2010.08.002. [DOI] [PubMed] [Google Scholar]
  58. Kunihisa M., Fukino N., Matsumoto S. Development of cleavage amplified polymorphic sequence (CAPS) markers for identification of strawberry cultivars. Euphytica. 2003;134:209–215. [Google Scholar]
  59. Kunihisa M., Fukino N., Matsumoto S. CAPS markers improved by cluster specific amplification for identification of octoploid strawberry (Fragaria × ananassa Duch.) cultivars, and their disomic inheritance. Theor. Appl. Genet. 2005;110:1410–1418. doi: 10.1007/s00122-005-1956-1. [DOI] [PubMed] [Google Scholar]
  60. Lande R. Genetics and demography in biological conservation. Science. 1988;241:1455–1460. doi: 10.1126/science.3420403. [DOI] [PubMed] [Google Scholar]
  61. Lehmann C. Clonal diversity of populations of Caiamagrostis epigejos in relation to environmental stress and habitat heterogeneity. Ecography. 1997;20:483–490. [Google Scholar]
  62. Levinson G., Gutman G.A. Slipped-strand mispairing: a major mechanism for DNA sequence evolution. Mol. Biol. Evol. 1987;4:203–221. doi: 10.1093/oxfordjournals.molbev.a040442. [DOI] [PubMed] [Google Scholar]
  63. Liu W.X., Liu W.H., Wu J., Gao A.N., Li L.H. Analysis of genetic diversity in natural populations of Psathyrostachys huashanica keng using microsatellite (SSR) markers. Agri. Sci. China. 2010;9(4):463–471. [Google Scholar]
  64. Liu Y.G., Liu L.X., Wang L., Gao A.Y. Determination of genetic stability in surviving apple shoots following cryopreservation by vitrification. CryoLetters. 2008;29:7–14. [PubMed] [Google Scholar]
  65. Liu Y.L., Li Y.H., Zhou G.A., Uzokwe N., Chang R.Z., Chen S.Y., Qiu L. Development of soybean EST-SSR markers and their use to assess genetic diversity in the subgenus Soja. Agri. Sci. China. 2010;9(10):1423–1429. [Google Scholar]
  66. Liu Z.Z., Guo R.H., Zhao J.R., Cai Y.L., Wang F.G., Cao M.J., Wang F.G., Rong H., Shi Y.S., Song Y.C., Wang T.Y., Li Y. Analysis of genetic diversity and population structure of maize landraces from the South maize region of China. Agri. Sci. China. 2010;9(9):1251–1262. [Google Scholar]
  67. Martin C., Cervera M.T., Gonzalez-Benito M.E. Genetic stability analysis of Chrysanthemum (Chrysanthemum x morifolium Ramat) after different stages of an encapsulation–dehydration cryopreservation Protocol. J. Plant Physiol. 2011;168:158–166. doi: 10.1016/j.jplph.2010.06.025. [DOI] [PubMed] [Google Scholar]
  68. Meneghetti S., Costacurta A., Frare E., Rold G.D., Migliaro D., Morreale G., Crespan M., Sotes V., Calo A. Clones identification and genetic characterization of garnacha grapevine by means of different PCR-derived marker systems. Mol. Biotechnol. 2011;48:244–254. doi: 10.1007/s12033-010-9365-3. [DOI] [PubMed] [Google Scholar]
  69. Moghaddam M., Omidbiagi R., Naghavi M.R. Evaluation of genetic diversity among Iranian accessions of Ocimum spp. using AFLP markers. Biochem. Syst. Ecolol. 2011;39:619–626. [Google Scholar]
  70. Mondini L., Noorani A., Pagnotta M.A. Assessing plant genetic diversity by molecular tools. Diversity. 2009;1:19–35. [Google Scholar]
  71. Morgana C., Di Lorenzo R., Carimi F. Somatic embryogenesis of Vitis vinifera L. (cv. Sugraone) from stigma and style culture. Vitis. 2004;43:169–173. [Google Scholar]
  72. Moslemi M., Zahravi M., Khaniki G.B. Genetic diversity and population genetic structure of pomegranate (Punica granatum L.) in Iran using AFLP markers. Sci. Hort. 2010;126:441–447. [Google Scholar]
  73. Munoz-Falcon J.E., Vilanova S., Plazas M., Prohens J. Diversity, relationships, and genetic fingerprinting of the Listada de Gandía egg plant landrace using genomic SSRs and EST-SSRs. Sci. Hort. 2011;129:238–246. [Google Scholar]
  74. NCWCD., 1998. Species status and conservation strategy. B. Endangered, vulnerable and rare plant taxa in the Kingdom of Saudi Arabia. National Commission for Wildlife Conservation and Development. Riyadh.
  75. Nemri A., Neff M.M., Burrell M., Jones J.D.G., Studholme D.J. Marker development for the genetic study of natural variation in Arabidopsis thaliana. Bioinformatics. 2007;23:3108–3109. doi: 10.1093/bioinformatics/btm501. [DOI] [PubMed] [Google Scholar]
  76. Nybom H. Comparison of different nuclear DNA markers for estimating intraspecific genetic diversity in plants. Mol. Ecol. 2004;13:1143–1155. doi: 10.1111/j.1365-294X.2004.02141.x. [DOI] [PubMed] [Google Scholar]
  77. Nybom H., Bartish I.V. Effects of life history traits and sampling strategies on genetic diversity estimates obtained with RAPD markers in plants. Perspect. Plant Ecol. Evol. Syst. 2000;3:93–114. [Google Scholar]
  78. Obembe O.O., Popoola J.O., Leelavathi S., Reddy S.V. Advances in plant molecular farming. Biotechnol. Adv. 2011;29:210–222. doi: 10.1016/j.biotechadv.2010.11.004. [DOI] [PubMed] [Google Scholar]
  79. Onay A., Pirinç V., Işıkalan C., Adıyaman F., Tilkat E., ve Başaran D. In vivo and in vitro micrografting of pistachio, Pistacia vera L.cv.“Siirt”. Tur. J. Biol. 2003;27:95–100. [Google Scholar]
  80. Paran I., Michelmore R.W. Development of reliable PCR based markers linked to downy mildew resistance genes in lettuce. Theor. Appl. Genet. 1993;85:985–993. doi: 10.1007/BF00215038. [DOI] [PubMed] [Google Scholar]
  81. Peirce L.C., Brewbaker J.L. Applications of isozyme analysis in horticultural science. Hort. Sci. 1973;8:17–22. [Google Scholar]
  82. Pernisova M., Klima P., Horak J., Valkova M., Malbeck J., Soucek P., Reichman P., Hoyerova K., Dubova J., Friml J. Cytokinins modulate auxin-induced organogenesis in plants via regulation of the auxin efflux. Proc. Nat. Acad. Sci. USA. 2009;106:3609–3614. doi: 10.1073/pnas.0811539106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  83. Popova E., Kim H.H., Paek K.Y. Cryopreservation of coriander (Coriandrum sativum L.) somatic embryos using sucrose preculture and air desiccation. Hortic. Sci. 2010;124:522–528. [Google Scholar]
  84. Prado M.J., Grueiro M.P., Gonzalez M.V., Testillano P.S., Dominguez C., Lopez M., Rey M. Efficient plant regeneration through somatic embryogenesis from anthers and ovaries of six autochthonous grapevine cultivars from Galicia (Spain) Sci. Hort. 2010;125:342–352. [Google Scholar]
  85. Rahman M.A., Mossa J.S., Al-Said M.S., Al-Yahya M.A. Medicinal plant diversity in the flora of Saudi Arabia 1: a report on seven plant families. Fitoterapia. 2004;75:149–161. doi: 10.1016/j.fitote.2003.12.012. [DOI] [PubMed] [Google Scholar]
  86. Reed B.M. In: Plant Cryopreservation: A Practical Guide. Reed B.M., editor. Springer; 2008. p. 406. [Google Scholar]
  87. Reed D.H., Frankham R. Correlation between fitness and genetic diversity. Conserv. Biol. 2003;17(1):230–237. [Google Scholar]
  88. Sanchez C., Martinez M.T., Vidal N., San-Jose M.C., Valladares S., Vieitez A.M. Preservation of Quercus robur germplasm by cryostorage of embryogenic cultures derived from mature trees and RAPD analysis of genetic stability. Cryo. Lett. 2008;29:493–504. [PubMed] [Google Scholar]
  89. Sarwat M., Das S., Srivastava P.S. AFLP and SAMPL markers for characterization of genetic diversity in Terminalia arjuna: a backbone tree of Tasar silk industry. Plant Syst. Evol. 2011;293:13–23. [Google Scholar]
  90. Schonewald-Cox C.M., Chambers S.M., MacBryde B., Thomas W.L. Benjamin/Cummings; Menlo Park, CA: 1983. Genetics and Conservation: A Reference for Managing Wild Animals and Plant Populations. [Google Scholar]
  91. Singh A., Chaudhury A., Srivastava P.S., Lakshmikumuran M. Comparison of AFLP and SAMPL markers for assessment of intra-population genetic variation in Azadirachta indica A. Juss. Plant Sci. 2002;162:17–25. [Google Scholar]
  92. Singh B., Sharma S., Rani G., Hallan V., Zaidi A.A., Virk G.S., Nagpal A. In vitro micro-grafting for production of Indian citrus ring spot virus (ICRSV)-free plants of kinnow mandarin (Citrus nobilis Lour × C. deliciosa Tenora) Plant Biotechnol. Rep. 2008;2(2):137–143. [Google Scholar]
  93. Sivakumar G. Bioreactor technology: a novel industrial tool for high-tech production of bioactive molecules and biopharmaceuticals from plant roots. Biotechnol. J. 2006;1(12):1419–1427. doi: 10.1002/biot.200600117. [DOI] [PubMed] [Google Scholar]
  94. Skoog F., Miller C.O. Chemical regulation of growth and organ formation in plant tissue cultures in vitro. Symp. Soc. Exp. Biol. 1957;11:118–131. [PubMed] [Google Scholar]
  95. Spaniolas S., May S.T., Bennett M.J., Tucker G.A. Authentication of coffee by means of PCR-RFLP analysis and lab-on-a-chip capillary electrophoresis. J. Agric. Food Chem. 2006;54:7466–7470. doi: 10.1021/jf061164n. [DOI] [PubMed] [Google Scholar]
  96. Sreekumar V.B., Renuka C. Assessment of genetic diversity in Calamus thwaitesii BECC.(Arecaceae) using RAPD markers. Biochem. Syst. Ecol. 2006;34:397–405. [Google Scholar]
  97. Srivastava S., Srivastava A.K. Hairy root culture for mass-production of high-value secondary metabolites. Cr. Rev. Biotechn. 2007;27(1):29–43. doi: 10.1080/07388550601173918. [DOI] [PubMed] [Google Scholar]
  98. Tahan O., Geng Y., Zeng L., Dong S., Chen F., Chen J., Song Z., Zhong Y. Assessment of genetic diversity and population structure of Chinese wild almond, Amygdalus nana, using EST- and genomic SSRs. Biochem. Syst. Ecol. 2009;37:146–153. [Google Scholar]
  99. Tanaka M., Takahata Y., Nakayama H., Yoshinaga M., Kumagai T., Nakatani M. Development of cleaved amplified polymorphic sequence (CAPS)-based markers for identification of sweet potato cultivars. Sci. Hort. 2010;123:436–442. [Google Scholar]
  100. Tianniam S., Bamba T., Fukusaki E. Pyrolysis GC–MS-based metabolite fingerprinting for quality evaluation of commercial Angelica acutiloba roots. J. Biosci. Bioeng. 2010;109(1):89–93. doi: 10.1016/j.jbiosc.2009.06.025. [DOI] [PubMed] [Google Scholar]
  101. Troggio M., Malacarne G., Coppola G., Segala C., Cartwright D.A., Pindo M., Stefanini M., Mank R., Moroldo M., Morgante M., Grando M.S., Velasco R. A dense single-nucleotide polymorphism-based genetic linkage map of grapevine (Vitis vinifera L.) anchoring Pinot Noir bacterial artificial chromosome contigs. Genetics. 2007;176:2637–2650. doi: 10.1534/genetics.106.067462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  102. Viana e Souza H.A., Lovato M.B. Genetic diversity and structure of the critically endangered tree Dimorphandra wilsonii and of the widespread in the Brazilian Cerrado Dimorphandra mollis: implications for conservation. Biochem. Syst. Ecol. 2010;38:49–56. [Google Scholar]
  103. Vicente MJ., Segura F., Aguado M., Migliaro D., Franco J.A., Martinez-Sanchez J.J. Genetic diversity of Astragalus nitidiflorus, a critically endangered endemic of SE Spain, and implications for its conservation. Biochem. Syst. Ecol. 2011;39(3):175–182. [Google Scholar]
  104. Vijayan K., Saratchandra B., Teixeira da Silva J.A. Germplasm conservation in mulberry (Morus spp.) Sci. Hort. 2011;128:371–379. [Google Scholar]
  105. Wang C., Zhang H., Qian Z.Q., Zhao G.F. Genetic differentiation in endangered Gynostemma pentaphyllum (Thunb.) Makino based on ISSR polymorphism and its implications for conservation. Biochem. Syst. Ecol. 2008;36:699–705. [Google Scholar]
  106. Wang C.N., Moller M., Cronk Q.C.B. Population genetic structure of Titanotrichum oldhamii (Gesneriaceae), a subtropical bulbiliferous plant with mixed sexual and asexual reproduction. Ann. Bot. 2004;93:201–209. doi: 10.1093/aob/mch028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  107. Wang S., Yang W., Shen H. Genetic diversity in Apium graveolens and related species revealed by SRAP and SSR markers. Sci. Hort. 2011;129:1–8. [Google Scholar]
  108. Welsh J., McClelland M. Fingerprinting genomes using PCR with arbitrary primers. Nucl. Acids Res. 1990;18(24):7213–7218. doi: 10.1093/nar/18.24.7213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  109. Williams J.G.K., Kubelik A.R., Livak K.J., Rafalski J.A., Tingey S.V. Nucl. Acids Res. 1990;18:6531–6535. doi: 10.1093/nar/18.22.6531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  110. Wongniam S., Somana J., Swangpol S., Seelanan T., Chareonsap P., Chadchawan S., Jenjittikul T. Genetic diversity and species-specific PCR-based markers from AFLP analyses of Thai bananas. Biochem. Syst. Ecol. 2010;38:416–427. [Google Scholar]
  111. Wu Y.G., Guo Q.S., He J.C., Lin Y.F., Luo L.J., Liu G.D. Genetic diversity analysis among and within populations of Pogostemon cablin from China with ISSR and SRAP markers. Biochem. Syst. Ecol. 2010;38:63–72. [Google Scholar]
  112. Xie G.W., Wang D.L., Yuan Y.M., Ge X.J. Population genetic structure of Monimopetalum chinense (Celastraceae), an endangered endemic species of Eastern China. Ann. Bot. 2005;95:773–777. doi: 10.1093/aob/mci087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  113. Xie W.G., Zhang X.Q., Cai H.W., Liu W., Peng Y. Genetic diversity analysis and transferability of cereal EST-SSR markers to orchardgrass (Dactylis glomerata L.) Biochem. Syst. Ecol. 2010;38:740–749. [Google Scholar]
  114. Yang M., Zhang X., Liu G., Yin Y., Chen K., Yun Q., Zhao D., Al-Mssallem I.S., Yu J. The complete chloroplast genome sequence of date palm (Phoenix dactylifera L.) PLoS ONE. 2010;5(9):e12762. doi: 10.1371/journal.pone.0012762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  115. Yao H., Song J.Y., Ma X.Y., Liu C., Li Y., Xu H.X., Han J.P., Duan L.S., Chen S.L. Identification of Dendrobium species by a candidate DNA barcode sequence: the chloroplast psbA-trnH intergenic region. Planta Med. 2009;75:667–669. doi: 10.1055/s-0029-1185385. [DOI] [PubMed] [Google Scholar]
  116. Zhang H., Mao X., Wu X., Wang C., Jing R. An abiotic stress response gene TaSnRK2.7-B in wheat accessions: genetic diversity analysis and gene mapping based on SNPs. Gene. 2011;478:28–34. doi: 10.1016/j.gene.2011.01.011. [DOI] [PubMed] [Google Scholar]
  117. Zhang Y.B., Shaw P.C., Sze C.W., Wang Z.T., Tong Y. Molecular authentication of Chinese herbal materials. J. Food Drug Anal. 2007;15(1):1–9. [Google Scholar]
  118. Zhou G., Yang L., Li C., Xu W., Chen G. Genetic diversity in endangered Notopterygium forbesii Boissieu based on intra-species sequence variation of chloroplast DNA and implications for conservation. Biochem. Syst. Ecol. 2010;38:911–916. [Google Scholar]
  119. Zhu S., Bai Y., Oya M., Tanaka K., Komatsu K., Maruyama T., Goda Y., Kawasaki T., Fujita M., Shibata T. Genetic and chemical diversity of Eleutherococcus senticosus and molecular identification of Siberian ginseng by PCR-RFLP analysis based on chloroplast trnK intron sequence. Food Chem. 2011;129(4):1844–1850. [Google Scholar]
  120. Zietkiewicz E., Rafalski A., Labuda D. Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification. Genomic. 1994;20:176–183. doi: 10.1006/geno.1994.1151. [DOI] [PubMed] [Google Scholar]
  121. Zuo Y., Chen Z., Kondo K., Funamoto T., Wen J., Zhou S. DNA barcoding of Panax species. Planta Med. 2010;77:182–187. doi: 10.1055/s-0030-1250166. [DOI] [PubMed] [Google Scholar]
  122. Zuzarte M.R., Dinisa A.M., Cavaleiro C., Salgueiro L.R., Canhoto J.M. Trichomes, essential oils and in vitro propagation of Lavandula pedunculata (Lamiaceae) Ind. Crop Prod. 2010;32:580–587. [Google Scholar]

Articles from Saudi Journal of Biological Sciences are provided here courtesy of Elsevier

RESOURCES