The Lamiaceae Family Mint Biology Essay

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Lamiaceae (syn. Labiatae) also known as the mint family is one of the large plant families (Wink, 2003). It is an important family containing close to 252 genera and around 6700-7000 species (Hedge, 1992; Mabberley, 1997). Several researchers described the Lamiaceae family as being mostly known for the wealth of species with enormous medicinal virtues. They are mostly aromatic plants comprising herbs such as sage, thyme, basil, mint, marjoram, rosemary and perilla (Wink, 2003; Celiktas et al., 2007; Hussain et al., 2008). Some of them are shrubs; a few are vines or trees. Many of these species for examples: Mentha arvensis, M. piperita, Ocimum basilicum, O.sanctum, Thymus vulgaris, T. linearis are found in different parts of the world being cultivated as commercial crops but are mostly found growing around the Mediterranean region (Wazir et al., 2004; Hussain et al., 2008; Hussain et al., 2010). The other species grow wild in mountainous region.

The plants have stems which are square in shape and having decussate leaves which are often hairy, billabiate flowers and four stamens (Two longer and two shorter). Flowers are symmetrical with five united sepals and five united petals. They are mostly bisexual and verticillastrate having one style found between the lobes of the ovary, fruit and one seeded nutlet forming two carpels within the calyx (Catino et al., 1992; Hoywood et al., 2007).

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The family is best known also a source of aroma compounds and essential oils which are used against different diseases and disorder and as antibacterial additives (Burt, 2004). Toxic compounds have been described in some Lamiaceae species yet these plant species are grown and explored for their leaves and oils for more than 75 years. The family is also famous for the presence of diterpenoids in its members.

Members of the family have a variety of economic uses and are rich of biodiversity. Since early times species of the Lamiaceae are being used by humans in culinary species and traditional medicine. They are also used for many purposes in areas such as ornamental, perfume industry, flavorings, vegetables and in other industry (Box, 1982; Jaleel et al., 2008). Other traditional uses cited by Mulas, (2006) are to combat pests and parasites, producing soap, for liqueur production, livestock feeding, house decoration, as well as fuel, alcoholic infusion and ointment.

1.2 THE GENUS OCIMUM

Ocimum L. collectively known as basil (Simo et al., 1990) belongs to the subfamily Nepetoideae and is ranked high among the other herbs. It is an important economic herb well known for their medicinal properties and cultivated for their essential oils but yet its taxonomy is really confusing as same species may be referred to as more than one name not following The International Code Of Botanical Nomenclature (Greuter et al., 1994) where names are attached by a certain rules and given a specific name of recognition.

The state of puzzlement is due to regular occurrence of the interspecific hybridization, polyploidization and races with related morphological characteristics and traits within the genus. Uncertainty in an accurate classification depends on the species identification also which are affected by developmental and environmental factors creating taxonomy challenges and differences leaving very little publications on Basil taxonomy which takes into account The International Code of Botanical nomenclature (A.J Paton et al., 2002). Based on extensive taxonomical studies variation of species may be from 30 (Paton, 1992) to 160 (Pusphpangadan and Brava, 1995) depending on several reasons like geographical and generic description. Estimates of species number differ in literature according to different research done by several authors.

Recent study comprises more than 150 species growing widely on the temperate regions all around the world. (Martin et al., 1999; Jirovetz and Buchbauer, 2001; Mondal et al., 2007). Most common species are Ocimum tenuiflorum (syn O.sanctum) O. gratissimum and O. basilicum, are known as holy basil, clove basil or wild basil and sweet basil respectively (Silva et al., 2004; Zheljazkov et al., 2008). We also have Ocimum americanum known as hairy basil and a few hybrids Ocimum Ã- citriodorum and Ocimum kilimandscharicum Ã- basilicum named lemon Basil and Dark Opal respectively. Chromosome number in Ocimum species is x=12 ((Carovic-Stanko et al., 2010) but Pushpangadan et al. (1975) reported that Ocimum basilicum is tetraploid and frequently has 2n=2x=48 (Xing-Hua et al., 1984; Ryding, 1994). It has been noted that though Ocimum has so much medicinal properties and is so much of economic importance, it has poorly interested researchers and this can be testified by the very few experiments only performed to date.

1.3 Botanical Description of Ocimum

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Members of this genus can be annual or perennial herbs and shrubs. They are strongly aromatic plants which do not have a rhizome and possess only a tap root. The leaves are deciduous with gland dots, can be petiolate or sessile toothed at the margin. Flowers are zygomorphic with two distinct lips. Typical inflorescence is composed two opposing cymes known as vertical.

The calyx is 5-lobed with an upper posterior single-lobed lip and a 4-lobed lower anterior lip of 2 lateral and 2 median lobes and their shape and position vary. The lateral lobes are found midway between the median lobes of the anterior lip and the posterior lip. They can be lanceolate and symmetrical or asymmetric. The posterior lip is decurrent forming inconspicuous or prominent wings. The calyx is short and tubular in shape and it can sometime be funnel shaped, straight or slightly curved. It often enlarges a bit in fruit. The throat is open and glabrous or bearded with a sphere of hair just below the mouth.

The corolla is straight and can be curved slightly to a downward position. At the midpoint the corolla tube is dorsally gibbous opposite the appendage of the posterior stamen. The tube dilates parallel-sided towards the mouth. The posterior lip of the corolla is always 4-lobed, but the lobes can be equal or unequal depending on species. The anterior lip is entire and usually horizontal, but sometimes becomes deflexed.

The Androecium have 4 stamens, one anterior pair attach near the corolla mouth where the stamens are usually free or sometimes fused together and a posterior pair which attach near the corolla base. The bases of posterior stamen are usually pubescent or glabrous. According to literature all stamens are fertile but we do have an exception where the posterior stamens are infertile like in O.drdnatum. The anther is two locular and dorsified which can be parallel or divergent, equidistant or unequal in lenth depending on species.

The ovary is divided into four parts in all species which develop into one single seeded nutlet or mericarps. The nutlets are spherical, glabrous and can produce copious mucilage when wet. The style is gynobasic and bifid with subulate or rounded lobes.

1.4 SPECIES DESCRIPTION

The species under investigation are Ocimum tenuiflorum (syn O. sanctum) and O. basilicum. They are described below.

1.4.1 Ocimum tenuilforum (syn O. Sanctum)

Table 1. Scientific Classification of Ocimum tenuiflorum

Botanical Classifcation

Kingdom

Plantae

Division

Magnoliophyta

Classs

Magnoliopsida

Order

lamiales

Famliy

labiatae

Genus

Ocimum

Species

sanctum

Source: http://www.ayushveda.com/herbs/ocimum-sanctum.htm

Ocimum Sanctum (syn O. tenuiflorum) considered as a sacred plant is commonly known as Holy Basil or 'Tulsi' (The Queen of Indian Herbs or The Mosquito Plant in English) and is very renowned for its miraculous medicinal qualities and for its spiritual sanctity. It is native from India and is also originates from some Africa and Asian regions. This plant is the most prominent species of this genera cultivated in many tropical parts of the globe. Tulsi plant is an erect, much branched, softy hairy herbaceous annual herb with thin simple opposite with varying in colors and with flowers white to purple (Gurib Fakim, 2007). Several botanists identified three main forms of Tulsi in cultivation (Maimes, 2004). Tulsi has great variation among the several domesticated cultivars. Ocimum Sanctum, has two varieties known as Sri tulsi (Ocimum sanctum = O. tenuiflorum) with stems and leaves of green or bright green, and one with deep purple leaves known as Krishna tulsi (Ocimum sanctum = O. tenuiflorum) with stems and sometimes also leaves of dark green to deep purple. Vana tulsi (Ocimum gratissimum), is an unmodified wild form of tulsi found in the forest. In Mauritius, Ocimum sanctum is known as

Purple Tulsi which has puple stem and leaves sometimes being deeper in color and Ocimum tenuiflorum as White Tulsi has light to green leaves. A few pictures of the different varieties of both White and Purple Tulsi taken at the NPPO station reduit to demonstrate the differences between the species.

( a) (b)

Figure 1. White Tulsi leaves in the isolation of DNA. (a) Purple stems and soft green leaves; (b) bright green leaves.

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(b)

Figure 2. Purple leaves which have been used of DNA isolation process. (a) Purple leaves; (b) deep purplish leaves.

All the varieties though being different have common medicinal properties. It is widely cultivated as all parts of the plants have useful application. Holy basil is known for thousands years since prehistoric times in the world for medicinal virtues and has gain momentum mostly after the 20th century and are widely used in Ayurvedic system despite this fact being a powerful Ayurvedic herb, it is still not so popular in the western world. It also has some major components which are tannins in 4.6% and essential oil 2% and this oil has several other components such as eugenol, caryophyllene and methyl chavicol and linaloll (Kothari et al., 2004). These components have insecticidal and anti-bacterial properties against mosquitoes and insects and also inhibit growth of Mycobacterium tuberculosis. The essential oil are used in curing many diseases such as skin diseases, hepatic disorders, cold, cough, malarial fever (Prakash and Gupta, 2005; Hannan et al., 2006; Muthuraman et al., 2008). By extraction process a juice is obtain from the leaves which has diaphoteretic, antiperiodic and expectorant properties. Tulsi promotes cardiovascular health, protects the body at cellular level, protects against unwanted bacterial growth and cures from respiratory disease like cold, cough and congestions. Tulsi also reduces respiratory problems like asthma and is also an immune booster.

1.4.2 Ocimum Basilicum

Table 2. Scientific Classification of Ocimum basilicum

Botanical Classification

Kingdom

Plantae

Phylum

Magnoliophyta

Class

Magnoliopsida

Order

Lamiales

Family

Labiatae

Genus

Ocimum

Species

Basilicum

Ocimum basilicum, commonly known as basil or sweet basil is originally native to India but is of cosmopolitan distribution mostly found in tropics and warm areas in Africa and Asia and also growing wild in some Pacific Islands. The two main types of cultivars mostly found in Mauritius are the sweet basil having green leaves and the Dark Opal with deep purple leaves as shown in the figures below. Basil is a pleasant tender growing herb with green leaves, almost hairless, it can be annual or perennial (Wister, 1991). It is the most popular aromatic plants having strong pungent and sweet smell. There are more than 60 varieties of basil which differ in appearance and taste from one another. Basil can grow up to 20-60cm long and is very popular for its pharmaceutical, culinary and essential oil properties (C.Gopi et al., 2005). It is widely used mostly in Far East regions and form an integrated part of the Asian cuisines cultivated for years to flavor foods. Basil is a worldwide economic crop well known for its essential oil properties (Grayer et al., 1996; Begum et al., 2002). Its oil production is 100 tonnes per year (Begum et al., 2002) and is the most widespread oil currently being produced in large amount in countries like India, Egypt, Pakistan, Comores, Reunion, Madagascar and Bulgaria (Lawrence, 1992). It has several chemotypes or cultivars such as lemon basil, anise basil and cinnamon basil with different oil composition due to polymorphism varying in flower color and leaf structure which can be from small and liniform to large and round (Hassagawa et al., 1997). According to Lawrence (1992), basil plant morphology variation is remarkable along with its oil composition. Being medicinal herb basil is also used for treating diarrhea, coughs, headaches and kidney malfunctions. Moreover the essential of basil has antifungal, physiochemical and insect repelling activity (Lahariya and Rao, 1979; Dube et al., 1989; özcan, 1998; Martins et al., 1999) as well as nematocidal and antimicrobial activity (Hili et al., 1997) and some inhibitory effect on Aspergillus Orchralus (Basilico and Basilico, 1999). The plant is stomatic, diaphoretic, carminative and a stimulant (Sahoo et al., 1997; Phippen and Simon, 2000). According to literature Ocimum basilicum is said to be closely related to and frequently confused with Ocimum africanum and Ocimum americanum, but they can be identified on the basis of hair distribution and flower size where Ocimum basilicum is almost hairless. Lemon-scented cultivars are usually the result of crosses between O. basilicum and O. africanum. Some other application of the basil oil can be flavoring of confectionery and baked goods condiments, salad dressing, non alcoholic beverages, ice creams. Other uses can be in perfumery, as well as in dental and oral products.

(a) (b)

Figure 3. Basil leaves used for isolation of DNA. (a) Sweet basil with green leaves; (b) Dark Opal with dark purplish leaves. Source for (b): http://www.edenbrothers.com/store/basil_seeds_dark_opal.html

1.5 Nutritional Profile of Holy basil 'Tulsi' and Sweet basil

Holy basil as well as the Sweet basil is extremely rich in Vitamin K. and in minerals like Calcium and Iron. It a good source of dietary fiber having small amounts of Manganese, Potassium and Magnesium. In addition, it has good amount of Vitamin A and Vitamin C and small amount of Folate. They contain no caffeine and other stimulant. Calorie content of Holy or Sweet basil can be 23 calories in 100g of the plant. Calories from fat are 5.

Figure : Showing nutritional values of dried, ground basil (Food Rating System)

Source: http://www.whfoods.com/genpage.php?tname=foodspice&dbid=85

1.6 The DNA Extraction process

Medicinal and aromatic plants efficient conservation, management and utilization of the genetic diversity and assessment are gaining global momentum. The techniques are morphology, biochemical and more recent one the DNA technology. Crop improvement facilitated by modern technology is recognized as a key strategy for achieving food security and sustainable agriculture (Hautea and Escaler, 2004). These advent genomic based techniques to crop development provide an opportunity for making significant genetic advance in Tulsi (basil) plants. Researchers are studying all varieties in the germplasm in order to cope with the Ocimum crops improvement with respect to high productivity eliminating disorder and diseases (Vasanthaiah et al., 2007). To identify and characterize cultivar, application of DNA technology is playing a major role in agricultural research which has progressed tremendously over the last decades (Nybom, 1990). DNA based techniques are being widely used to assess genetic diversity and differences within species.

Isolation of a workable germplasm nucleic acids for analytical study and assessment using molecular techniques is the most crucial, time consuming steps (Puchoa, 2004) and challenging step in constructing of genomic library (Kit et al., 2010). Degree of purity and quality DNA varies between applications.

The isolation process involves firstly the mechanical breakdown of the cell wall in presence of liquid nitrogen allowing access to nuclear material , protein and other contaminants (Sun, 1994). The second step is the disruption of the cell membrane using CTAB and SDS detergents. The CTAB carrying a net charge is a cationic detergent solubilising membranes (Sghaier et al., 2005) interacts with the negatively charged DNA molecules forming a soluble stable complex of nucleic acid (Murray and Thompson, 1980). According to Foster and Twell (1996), this can be precipitated by lowering the salt concentration. The released DNA should be protected from endogenous nucleases. Thus, EDTA is included in the extraction buffer to chelate the Mg2+ ions, co-factor for nucleases action. This addition further helps in breaking the plant cells as the EDTA binds to Ca2+ and Mg2+ weakening the cell membrane stability (Puchoa, 2004).

A DNA extract is obtained which contain high level of secondary metabolites like tannins and alkaloids which contribute to its valuable medicinal virtues (Katterman and Shattuck, 1983), mainly polyphenolic compounds, contaminants, polysaccharides and large amount of RNA (Maria et al., 2001). Presences of these substances suppress and inhibit analysis of the isolated DNA and make separation process more difficult. Polyphenolic compounds makes DNA resistant against the restriction enzymes which directly or indirectly interfere with enzymes (Weishing et al., 1995) and interacts irreversibly by binding to protein and nucleic acids during homeginization process (Aljanabi et al., 1999; Manoj et al., 2007; Sharma et al., 2002).This process produces a gelatinous material which is hard and render the DNA unsuitable for PCR amplication and analyses. The polysaccharides inhibit the activity of the biological enzymes used such as polymerases ligases and restriction endonucleases (Sharma et al., 2002). Upon addition of alcohol the polysaccharides co-precipitate with DNA causing interference in quantification if nucleic acid by spectrometric methods (Wilkie et al., 1993). Removal of polysaccharides is done by increasing the NaCl concentration more than 0.5M increasing its solubility (Thompson and Murray, 1980). Proteins from the extract are removed by precipitation and denaturation using chloroform or phenol allowing separation of the organic and aqueous phases reducing foaming during the extraction process. To ensure that all proteins have been removed it is recommended to use chloroform: isoamyl alcohol several times. Precipitation is done with cold isopropanol and left overnight for further precipitation to occur. On the other hand RNAs chelate Mg 2+ reducing yield of PCR (Padmalatha and Prasad, 2000) and are generally remove by treatment of extract with heat treated RNASE A.

Antioxidants are commonly used to deal problems related to phenolic compounds examples include 2 β- mercapthoethanol, PVP, ascorbic acid (Dawson, 1995; Clark, 1997). 2 β- mercapthoethanol is used for preventing formation of phenolic substances produced by homogenization step where plant cells are broken to release nucleic acid and other complexes. PVP is unreactive and water soluble high molecular weight compound that binds with polyphenols through hydrogen bonds forming complexes (Maliyakal, 1992), however it should be noted that risk with PVP is that at a very high molecular weight it precipitates with the DNA and thus act as a contaminant (Puchoa and Khoyratty, 2004).

DNA is cleaned washed with 70% ethanol to remove salt contaminants that are readily soluble in ethanol solution and TE buffer is added to pellet the DNA.

All these problems encountered in isolation and purification process from medicinal and aromatic plants should be overcome by using a good DNA extraction protocol which will yield adequate and intact DNA of a reasonable purity. The process to be carried out should be easy, rapid and not making too much use of dangerous chemicals. Various protocols for isolating DNA have been successfully applied to many plant species (Dellaporta et al., 1983;; Roger and Benedich, 1985; Doyle and Doyle, 1990; Edwards et al., 1991; Ziegenhagen and Scholz, 1993). The only differences between these protocols are the ingredients pH and the extraction buffer. Each plant require its specific adjusting protocol depending on the demand of the level of the of DNA of the level of DNA purity. Biochemical composition of species shows much variation thus even closely related species may require different isolation protocols (Weishing et al., 1995).

1.7 Polymerase Chain Reaction (PCR)

Developed by Kary Mulis in the mid 1980s the PCR method was a great technological breakthrough in genome analysis. PCR is a simple and ingenious enzymatic replication method to amplify molecule of DNA sequences by in vitro DNA synthesis (Dubey, 2006). Its principle is based on the replication of original DNA molecule by DNA polymerase enzyme doubling the number of DNA molecules. It is a widely used technique as it is rapid, inexpensive, and versatile and is easy to use (Saxena et al., 2005). Amplification is done through repeated cycles. DNA may not be pure but it should not contain contaminants.

1.7.1 Steps of Mechanisms of PCR

PCR involves 3 main steps in the amplification cycle which are temperature dependent:

Denaturation ( 95°C)

The DNA template is denatured by heating at 95°C to separate the double stranded DNA into two single strands

Primer Annealing

The temperature is lowered to 65°C to cool the reaction mixture allowing the primers to pair up with single the strand which will then be amplified.

Elongation (Primer Extension)

This process involves a rise of temperature to 72°C allowing the DNA polymerase enzyme to attach to the primer site and synthesize a new DNA strand by addition of nucleotides to the 3' end of each primer.

Figure 4. Representation of Polymerase reaction

Source: http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechPCR.shtml.

1.7.2 PCR Parameters

DNA Template

Short DNA fragments in small amount which are not highly purified are required for PCR. The degraded DNA is more readily amplified as it is denatured easily thus making primer attachment to happen at ease (Kocher and Wilson, 1995). Inhibitors such as EDTA or phenols should be excluded in the reaction mixture (Mcpherson et al., 1993).

Primers

Short pieces of DNA of 18- 30 bases long designed to flank region for amplification. Range of concentration should be from 0.1 to 1μM otherwise a high concentration can cause undesirable amplification of non targeted sequences (Sambrook, 1989).

DNA polymerase

Taq. DNA polymerase is isolated from hot spring thermostable bacterium Thermus aquaticus. It catalyses incorporation of dNTPs into DNA. Being heat tolerant and active at high temperature it is widely used in PCR as it is not destroyed at increasing temperature and annealing process is more specific and rapid (Taylor, 1991).

Mg 2+

The nucleotides forms complex with the magnesium ions which act as a co-factor for Taq polymerase activity to happen efficiently. Its concentration should be kept quite low around 1.5mM. The DNA template should not contain dNTPs, chelating agents and proteins as this can affect the reaction process.

dNTPs (Deoxynucleotide triophosphate)

The dNTPs are sources of energy and provides nucleosides for DNA synthesis. Same amounts of nucleotides should be added or else excess of dNTPs can prevent product formation causing premature termination of the DNA process.

Buffer

The buffer contains MgCl2 and Tris Hcl. Its concentration is between a pH range of 8.2- 9.0 at 25°C for suitable PCR amplification. The buffer provides a good environment for the DNA to polymerase activity to happen at this pH range as the polymerase activity declines at higher or lower pH range (Taylor, 1991).

Annealing temperature

There is an optimum temperature for annealing primers to target DNA. At low temperature 37°C, efficient amplification occur but undesirable priming process also happen and at higher temperature like 55°C amplification process increases but its efficiency decreases (Sambrook et al., 1989).

Cycle Number

Amplification relies on the concentration of the targeted DNA. Amount of the enzymes after 25-30 amplification cycles decreases.

1.8 Morphological Markers

Morphological markers such as plant height, leaf shape and color correspond to qualitative visual traits. They can be dominant or recessive as a result of mutation. These markers have a vital role in crop improvement and plant breeding programs management but are less desirable to use in the evaluation of genetic variability as they are largely influenced by environmental factors, mutation and expression of genes (Geneve et al., 1997). Some plants may undergo mutation due to several genes interaction to express a single trait making the interpretation more confusing and difficult. Evaluation of morphological characters in Ocimum species found in Mauritius were based on plant height, leaf length, leaf width, leaf color, shape of the leaf, growth habit, color and strength of stem, venation and color of mid rib.

1.9 Molecular Markers

Molecular markers also known as DNA markers are used for genetic conservation, diversity and assessment as well as cultivar identifaication (Vural and Dageri, 2009) in order to paliate the problems which are encountered when using morphological markers to evaluate variation and detect differences in species. Selection of useful markers is based on availability, time, precision degree, economic resource and the nature of the research to be carried out. Molecular markers are specific to a particular DNA segment. They are the most practical aspect of biotechnology (Kumar and Kumar, 2000) as they do not require intensive labour and work and allow large distribution of the markers.

1.9.1 Types of Molecular Markers

There are diverse types of DNA- based molecular techniques established to evaluate DNA polymorphism which include hybridization-based methods, PCR-based methods and sequencing methods (Joshi et al., 2004).

1.9.1.1 Hybridisation-based methods

This technique uses labeled probes to hybridise and filter the digested DNA with restriction enzymes. The methods include Restriction Fragment Length Polymorphism (RFLP), Minisatellites and Variable Number Tandem Repeats (VNTRs) (Joshi et al., 2004).

Restriction Fragment Length Polymorphism (RFLP)

This method uses RFLP probes which are widely used for genome mapping and to identify population and cultivar identification. These are co-dominant markers that can differentiate heterozygote from homozygote at species level (Dubey, 2005). Its shows high reproducibility and are very reliable but it is very costly and require great skills and is labour-intensive. The genomic DNA is digested with restriction enzymes to generated fragments (Aquadro et al., 1992). DNA is rearranged due to evolutionary processes where insertions and deletions within the fragments occur (Kumar and Kumar, 2000) giving rise to differences which can be analysed. However, complicated banding patterns may cause difficulties in the evaluation of results (Striem et al., 1990). An example where RFLP method has been used is for sugarcane genetic diversity (Holton, 2001).

Microsatellites or Simple Sequence repeats (SSRs)

Microsatellites are known as short variable tandem repeats consisting of short sequence motif nucleotides. They are ubiquitous in plant genomes (Lagercrantz et al., 1993; Wang et al., 1994) and are highly informative molecular probes which have wide applications in plant genetics (Morgant and Olivieri, 1993) as well as in areas of genotype fingerprinting and identification due to their highly polymorphic (Tautz, 1989) and multi allele nature. They are also locus specific, co-dominant with a large amount in genome (Gupta and Varshnay, 2000). This technique has successfully been mapped in several essential aromatic and medicinal crops (Tripathi et al., 2008, 2009) and other economic plants. A few examples among are rice (Cho et al., 1994) and wheat (Roder et al., 1998). This method is based on affinity capture of single-stranded restriction fragments which are annealed to biotinylated microsatellites base pairs and magnetic separation.

1.9.1.2 PCR-based Methods

These involve the in-vitro amplification of a particular DNA segment using specific or arbitrary chosen oligonucleotide primers and polymerase enzymes. The different methods are:

Amplified Fragments Length Polymorphism (AFLP)

AFLP is a polymerase chain reaction based on amplification of short selective restriction fragments of DNA onto which adaptors will be ligated at both ends. These markers are an economical tool, reliable, effective, and rapid having large genome coverage (Hansen et al., 1999; Prashanth et al., 2000). Due to their high efficiency and reproducibility (Vos et al., 2005) they are used for assessing genetic relationships (Belaj et al., 2003), to detect polymorphisms, construct genetic maps. AFLP markers are used to characterize genetic diversity in aromatic and medicinal plants including Ocimum (Labra et al., 2004; Carović-Stenko et al., 2010) to make some classifications about their inter-relationships.

Random Amplified Polymorphic DNA (RAPD)

RAPD markers are based on the amplification of DNA sequences with single, short and arbitrary oligonucleotide primers (Bardakci, 2001) selectively amplifying DNA sequences. RAPD techniques have drawn the attention and interest of many geneticists and scientists. These are simple and easily applicable markers to several species without the need of prior knowledge of DNA sequenece. Due to their simplicity and random distribution of genetic markers they are very useful for species genes identification (Paran et al., 1991); genetic mapping, studying close genetic relationships (Sang-Bok Lee et al., 2000) and gene expression pattern (Valle et al., 2000) in many economical crops. Some disadvantages of RAPD markers are its dominance, there is lack of cross transferability (Godwin et al., 2001) and sensitiveness of RAPD reaction to PCR reaction being short in length and may also include contaminant DNA from infections in the material the DNA is isolated. These related problems can be overcome by sequencing the fragments making them become sequence characterized amplified region which are more robust than RAPDs (Godwin et al., 2001).

1.9.2 Use of molecular markers in Lamiaceae family and Ocimum species

For genetic improvement of Ocimum genus only a limited molecular markers are available. Since there exist several medicinal species and varieties molecular markers can be a vital tool for quality assessment in pharmaceutical industry (Sang-Bok Lee et al., 2000). In the past years DNA markers have rapidly been integrated in tools for genome analysis of species in the Lamiaceae family. A few examples are the analysis of Mentha using AFLP markers by S. Moja et al. (2002), hybridization in Phlomis (Aparicio et al., 1999) and RAPD analysis of endemic Sideritis pusilla (Vasquez et al., 1998).

Viera et al. (1999) had analysed the genetic diversity of 12 Tree basil (O. gratissimum) accession using RAPD markers correlated to secondary products. Several other authors have used DNA markers (RAPD, RFLP, AFLP, SSR, and ISSR) to assess the genetic variability and studying phylogenetic relationships of species found in Lamiaceae family including Ocimum species. So far to my knowledge no use of RAPD markers in the genus of Ocimum had been reported in Mauritius.