Herbaceous Plants In The Genus Musa Biology Essay

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Banana is a common name for herbaceous plants in the genus Musa. Because of its structure and size; it is frequently mistaken as a tree. Bananas (closely linked to plantains) are belong to Eumusa section of the family Musaceae, and natural hybrid polyploidy (diploid, triploid or tetraploid) of two main species of Musa which are Musa acuminate, type A and Musa balbisiana, type B.

These tallest monocotyledons (Stover and Simmonds, 1987) have turn out to be one of the main significant fruit crops in the world providing most important export income as well as being a staple food for millions of citizens in many developing countries (FAO, 2002). Globally, bananas constitute the fourth most important food crop in human consumption following rice, wheat and maize. They are grown in more than 100 countries worldwide, more than for any other fruit crop. Bananas are inhabitant to tropical southeastern Asia. As a prime fruit commodity, bananas contribute significantly to the Asian diet and nutrition. In addition to its primary used as a dessert fruit and staple starch, bananas provide various secondary product for example premier fruit of Southeast Asia and is considered of great socioeconomic importance in the countries of the region. It ranks second or third in importance amongst the fruit industries of India, Malaysia and Taiwan. It is also one of the significant commodities for both domestic and export in Malaysia (Valmayor, 1987).

The International Network for the Improvement of Banana and Plantain (INIBAP) was created in 1985, becomes network of collections which handle with genetic resources of banana in all over the world. The basis of this network is provided by the gene bank of the INIBAP Transit Centre in Leuven, Belgium, where the bigger section of the banana gene pool is detained, in trust for humanity, in a tissue culture collection that serves as a 'safety back-up' for all the diversity of banana plants growing in field collections, farms and forests all over the world. Additional safety back-up is provided by a gradually rising collection of accessions that is held for the long period, with no deterioration, by 'cryopreservation' in liquid nitrogen (http://bananas.bioversityinternational.org).

Conventionally, people will recognize banana cultivars based on their morphological characters for example the leaves and fruit. However, morphological changes caused by environmental factors are the main obstacles to precisely identify the varieties (Kaemmer et al., 1992). Current research has conducted to assist the identification of bananas not only based on their morphology characters, but also using molecular genetics approach. This study is very practical to isolate and analyze genetic sequences of various kinds of banana species and based on that, the types of banana can be identified by study their molecular composition. Although this way of bananas identification is quite complex as compared to the morphological study, yet this kind of way can provide people with a new approach to recognize those bananas with higher confidence. In addition, this study is carrying out using banana plants simply because of its commercial value all over the world especially in Malaysia as well as its nutritional value.

Modern research has shown that most angiosperm species inherit their chloroplast maternally (Stegemann et al., 2003) not inherited from the male parent. Seeing as chloroplasts are inherited only from the female, transgenes in these plastids cannot be disseminated by pollen and posing extensively lower environmental risks. Certainly, chloroplast DNA can give precious information in population and phylogeny studies by choosing suitable DNA marker.

DNA markers have been established to be useful, efficient and consistent methods for genetic characterization, studying genetic diversity and relationships between populations and varieties since they are not exaggerated by environmental circumstances (William et al., 1990). Several DNA markers for bananas have been reported. These include Restriction Fragment Length Polymorphisms (RFLP) (Jarret et al., 1992; Ge et al., 2005), Random Amplified Polymorphic DNA (RAPD) (Onguso et al., 2004; Martin et al., 2006; Ray et al., 2006), microsatellite DNA (Creste et al., 2003; Buhariwalla et al., 2005) and Variable Number of Tandem Repeats (VNTR) polymorphisms (Kaemmer et al., 1992; Bhat et al., 1995). The used of rpoC as a genetic marker for this study was recommended by the Royal Botanic Gardens at Kew, London.

The aims of this study are:

To obtain the partial DNA sequences of rpoC.

To analyze either the rpoC gene can be used as a proper genetic marker for banana cultivars.

To study phylogenetic; analyze the genetic diversity, variation and inferring the relationship among selected local banana cultivars.



2.1 Origin and Distribution of Banana

Banana term is a common name referring to the species or hybrids belong to a genus Musa. Bananas, scientifically known as Musa spp. are perennial monocotyledonous herbs that grow healthy in humid tropical as well as subtropical regions. Bananas have an extensive diversity of historic references particularly in ancient Hindu, Chinese, Greek and Roman texts. The mainly primitive indication to banana dates back to about 500 BC. There are a number of scientists that involve in cultivating fruit, vegetables and other plants who think that banana was the former fruit on earth. Even so, banana's origin is traced back to Southeast Asia in the jungles of Malaysia, Indonesia and Philippines (Simmonds, 1966).

Banana was derived from two wild diploid (2n = 22) species specifically, Musa acuminata Colla and Musa balbisiana Colla, with genomic compositions of AA and BB, respectively (Cheesman, 1948). Musa accuminata is locally in Malay Peninsula and neighboring countries whereas Musa balbisiana is originate in India eastwards to the tropical Pacific (Simmonds, 1966). South East Asia and the Western Pacific are thought to be the main center of origin and domestication of edible bananas (Simmonds, 1962; Robinson, 1996; Jones, 2000), but they are broadly spread in the humid tropical and subtropical regions as well. From Asia, bananas and plantains are spread all the way through the humid tropics (Swennen and Ortiz, 1997; Valmayor, 2000) exclusively by man through suckers (Simmonds, 1962). Suckers spring up around the main plant forming a clump or "stool'', the earliest sucker replacing the main plant when it fruits and dies, and this course of action of succession continues for an indefinite period.

The historic times of banana cultivation is thus strongly related to the early progress of the human populations. Movement through eastwards resulted in the development of a distinct group of AAB bananas, which are cultivated throughout the Pacific Islands. On the other hand, during the 15th century, Arab traders from India were the one who introduced banana plants in Africa (Simmonds, 1962). The banana was then moved inwards by local migrants and soon after, from Africa it broadens to further parts of the tropical and subtropical regions. An enormous diversity of bananas and plantains currently present in sub-Saharan Africa by means of diverse types cultivated in different eco-regions (Swennen and Vuylsteke, 1991). The AAB type of the banana plantains present in the humid lowlands of West and Central Africa whereas the AAA type exists in the East African Highlands. The latter two regions represent the world's most diversity of plantains and highland bananas, correspondingly and therefore considered as the secondary centers of diversification of bananas and plantain (Swennen, 1990).

2.2 Banana Taxonomy

Banana is belongs to the genus Musa in the family Musaceae and order Zingiberales. It belongs to the subclass Zingiberidae, Class Liliopsida and Division Magnoliophyta. According to Cheesman (1948) and Simmonds (1962), the family Musaceae comprises two genera which are Ensete and Musa (Figure 1).

The first genus in the Musaceae family, Ensete consists of monocarpic herbs which they are not edible fruit. The genus was officially recognized by Cheesman in 1948 comprising 25 species. Yet only eight species are now recognized in the genus Ensete (Novak, 1992). Ensete is cultivated in Southern Ethiopia as a main supply of food that is obtained from the rhizome and also pseudostem (Novak, 1992). Only two species from this genus; E. ventricosum and E. edule, are of economic significance as food and fiber crops (Bezuneh and Feleke, 1966). Genetic associations flanked by Ensete species and Musa clones based on genome size, number of chromosomes and number of 45S rDNA loci showed that Ensete is strongly linked to M. beccarii of section Callimusa (Bartos et al., 2005).

The second genus, Musa comprises all edible bananas and plantains with more than 50 species. There are five sections exist in this genus which are Australimusa, Callimusa, Rhodochlamys, Eumusa and Ingentimusa (Stover and Simmonds, 1987; Purseglove, 1988). Those sections vary in the basic chromosomes, for example; species of Callimusa and Australimusa have a basic chromosome number of x = 10, while species in Eumusa and Rhodochlamys have a basic chromosome number of x = 11. Ingentimusa has a single species M. ingens with a chromosome number of 2n = 14. Eumusa is the largest, most far and wide distributed, extremely diversified and the most essential section to which all edible bananas belong. Nearly all cultivars in this section are derivative of two species, Musa acuminate (A genome) and Musa balbisiana (B genome). Musa acuminata is the most well-known of the Eumusa species being found throughout the range of the section with Malaysia (Simmonds, 1962) or Indonesia (Nasution, 1991) as the center of diversity.

Figure 2.2.1: Classification of Family Musaceae showing sectional treatment of the genus Musa. Source: Wil et al. (2001).

Figure 2.2.2: Important characters used in determining species and genome groups of edible bananas. Source: IBPGR Revised Banana Descriptors (1984).

Edibility and successive domestication of diploid M. acuminata (AA) came about as an outcome of female sterility and parthenocarpy. Triploid AAA cultivars arose from diploids, perhaps, following crosses among edible diploids and wild M. acuminata subspecies, giving rise to a wide range of diverse AAA genotypes such as the AAA dessert bananas and the AAA East African Highland bananas. These two AAA groups of bananas differ in their fruit morphology which considers to starch content and also the taste. This suggests that the A genomes of the dissimilar subspecies of Musa are different from each other. In the largest parts of South East Asia triploids have replaced the original AA diploids due to their larger fruit and vigorous growth. Nevertheless, in Papua New Guinea, AA diploids stay as an agriculturally significant and a broad diversity is still found in cultivation.

2.3 Morphology and Botanical Description of Banana

Banana plant is a huge perennial herb with a height ranging between 1.5 m to 10 m. It consists of a true stem called corm with roots and a very succulent (having fleshy tissues that conserve moisture) stem; well known as pseudostem which consisting of leaf-petiole sheaths, reaching a height between 6 m to 7.5 m and arising from a fleshy rhizome or a corm. Tender, smooth, oblong or elliptic, fleshy-stalked leaves with spirally arranged are numbering 4 to 15. When the plant grows, they are unfurling at the rate of one per week in a warm weather, and expand upward and outward, becoming as much as 2.75 m long and 60 cm wide. The stems grow to 4 m to 8 m tall, with large leaves 2 m to 3 m long. Bananas in general weigh between 125 g to 200 g, though this varies noticeably among different cultivars; of this, about 80% is edible, and the skin the remaining 20%, whereas a stem of bananas can weigh from 30 kg to 50 kg, and they are typically carried on the shoulder.

During maturity period, the leaves encircle the so-called "heart" that carries bunch together with the fruit. Corm, which is the stem is typically underground and its shape is cultivar dependent. Nevertheless, in nearly all cultivars the corm is round and the apical meristem is at its tip. The meristem will remains beneath the soil until flowering occurs when it develops into flower inflorescence axis that holds the bunch. Roots expand starting from the corm from the part between the inside zone (central cylinder) and the outside zone (cortex) into the soil. Leaves grow from the meristem of the corm as well and have a sheath, a petiole and lamina. The leaf sheaths of successive leaves lie on top and directly encircles each other forming pseudostem or the false stem. Highland and dessert bananas' pseudostems are green to dark green with many black blotches, whereas plantains are yellowish green with few brown-black blotches. As fresh and young leaves develop at the meristem, older leaves are pushed outwards, die and finally dry out (Simmonds, 1962). Nearly all bananas produce more or less 30 to 40 leaves in its life period.

As soon as enough number of leaves is produced, the meristem continues to become flowering stem, which starts to grow upwards through the pseudostem. The flowering stem emerges in the center of the leaf crown and a compound inflorescence of flower clusters develops. The female flowers become visible first and have big ovaries that develop into fruits afterward. When the inflorescence develops, a bulb formed male bud containing small flowers will develop at the last part. On the other hand, fruit of cultivated bananas develops by parthenocarpy, preventing growth of seeds that would or else make the fruit improper for human consumption.

Banana inflorescence produced three kinds of flowers. The female (pistillate) flowers will develop into fruit, whereas the male (staminate) flowers found in the male bud may produce pollen that may be fertile or sterile. The third type of flowers is called hermaphrodite or neuters which found on the inflorescence axis or rachis flanked by the female flowers and the male bud and they are generally sterile. The female flowers of most cultivated bananas are more usually sterile and the fruit develop by parthenocarpy.

In all bananas, the growing shoot dies soon after fruiting once (Simmonds, 1962) and its life is perpetuated by means of suckers, which grow from adventitious buds produced on the corm. The suckers are key form of vegetative planting material and form the following vegetative generation. As soon as the first plant fruits and dies, the maiden sucker continues the development cycle. Banana plants are propagated vegetatively through suckers, even though wild species can be propagated by seed as well (Stover and Simmonds, 1987). Sucker progress consists of three distinct stages; peeper (young sucker bearing scale leaves only), sword sucker (sucker bearing narrow sword leaves) and maiden sucker (large but non fruiting ratoon with foliage leaves) (Simmonds, 1966; Swennen et al., 1984). The cluster formed by the mother plant and the surrounding suckers is referred to as a 'mat'. The amount of suckers produced by a plant is essential to farmers in banana production as the crop is vegetatively propagated.

Banana cultivars can be found in variety of colors and sizes. Most cultivars are yellow once it ripe but some are red. The ripe fruit is can be peeled easily and eaten raw or cooked. Depends on cultivars and ripeness, the flesh can be starchy to sweet, and firm to mushy. Unripe or 'green' bananas are used in cooking and are the staple starch of several tropical populations.

As for human consumption, bananas that contain large seeds, seedless and triploid cultivars are selected. These are propagated asexually from offshoots of the plant. The plant is tolerable to produce 2 shoots at a time; a larger one for fruiting directly and a smaller 'sucker' or 'follower' that will produce fruit in about 8 months time. The life of a banana plantation is about 25 years or longer, throughout which time the individual stools or planting sites may move a little from their older positions as lateral rhizome formation dictates.

Cooking bananas grow best at altitudes around 1,200 to 1,800 meters above the sea level, while dessert bananas and plantains develop fine in the low lands as well as in deep loamy and well-drained soils. The optimum temperature for most cultivated bananas to grow is between 26 to 30°C (Stover and Simmonds, 1987). Temperatures lower than the optimum will affect leaf production, therefore decreasing the food supply due to the inadequate photosynthetic leaf area. Banana growth stops at temperatures above 38°C and will eventually dies at temperatures below 0°C. A relative humidity of 60% to 100% is essential for the banana to grow well and it is furthermore depending on the evapotranspiration; which 25 mm to 75 mm of water is needed by a banana plant per week which is equivalent to 100 mm rainfall per month. Bananas are prone to wind damage because of their weak pseudostems, large leaves that trap wind and have shallow root structure.

2.4 Major Areas of Production and Importance of Bananas

As stated by FAO (2002), bananas are the 4th world's most important food crop after rice, wheat and maize, with vast majority of the crop grown and consumed in the tropical and subtropical zones. Banana is more significant as food for local consumption than for export due to the annual world banana production is 98 million tons of which only 7 million tons go into the world market. The annual world banana production increase from 51 million tons to 88 million tons which about 70%, between the year 1970 and 1997 (Sharrock and Frisson, 1998). During those times, banana production was estimated to be growing faster than the production of any other starchy crop in the world. The world's current banana or plantain production is estimated at about 104 million metric tons, grown on about 10 million acres of land in over 100 countries (FAOSTAT, 2004).

In humid forest and mid altitude region of Africa, bananas provide around 25% of the carbohydrate supplies for more than 70 million people (Robinson, 1996), with per capita consumption of roughly about 250 kg. Its capacity to turn out fruit all the year makes it a significant food security crop and cash crop in the tropic regions (Jones, 2000). Bananas are prepared and consumed in various ways and each country that produces the crop having its own traditional dishes and ways of processing (Frison and Sharrock, 1998). As an example, mature unripe bananas are eaten as a starchy food whereas ripe bananas are consumed raw as a dessert fruit. Other than that, they can be consumed boiled, roasted, or fried in ripe or unripe condition as well. Nutritionally, fresh bananas contain 35% carbohydrates, 6-7% fiber, 1-2% protein and fat; further the main elements such as potassium, magnesium, phosphorus, calcium, iron, and vitamins A, B6 and C (Robinson, 1996). Bananas are also used in the manufacture of beer, wine and other products and for man important part of the cultural life of many people (Stover and Simmonds, 1987). Other products that can be produce by banana are like jam, juice and squashes, banana chip, sweet banana figs, banana flour, banana powder and starch. However unlike other fruits, bananas have historically been difficult to extract juice from because when they are compressed a banana can simply turns to pulp.

Bananas are valuable in prevention of cancer as well as heart diseases in humans due to their high vitamin A and B6 content. Bananas are also used to treat diseases such as gastric ulcer and diarrhea. Nectar sap from banana flower buds are rich in vitamins, so can be fed to babies and children to reinforce their growth and at the same time potassium helps in boosting brain performance.

Above and beyond the food and income, banana plays lots of vital roles. For example banana leaves can be used as thatching materials for houses, as plates, tablecloths, umbrellas, sleeping mats, animal feed and also in food preparation. Non-fruit parts of the banana plant, including the corm, shoots, pseudostem and male buds are eaten as vegetables in Africa and parts of Asia (Simmonds, 1962). Banana leaves and pseudostems are highly fiber which can be used as animal feed as well as for making ropes, handcraft, baskets, carpets and manufacturing of banana paper. In mixed cropping systems, banana plants provide shade for certain crops that grow better in shade conditions such as cocoa, black pepper, coffee and vanilla. At the system level, bananas keep up the soil structure and cover throughout the year, protecting it from wind and rain erosion. Additionally, if the biomass is used as mulch, soil fertility and organic matter remains stable.

In addition to the fruits, the flower of the banana plant is used in Southeast Asian, Bengali and Kerala (India) cooking, either served raw with dips or cooked in soups and curries. The gentle core of the banana trunk is also used in Burmese, Bengali and Kerala food preparation.

Banana leaves that generally large, flexible, and waterproof, are used as umbrellas and to wrap food for cooking purpose. In South India, the leaves are used as a natural plate to serve their food. Once eaten, the leaf is thrown away for cattle consumption, which is one example of an eco-friendly practice. This practice has regained popularity since it values hygiene as an important aspect and the fact that it saves on water and detergents that usually have been used to clean a plate. Furthermore, hot food served in a tender banana leaf manifests a distinct banana flavor that is also said to have nutritional benefits.

Bananas are sterile, meaning that they do not produce viable seeds. They are lacking seeds, so another form of propagation is needed. The two standard ways to plant bananas are to either transplant part of the root (called a "corm") or to transplant suckers (shoots that develop at the bases of the banana plant). Suckers are living plants and are too delicate to transport over long distances, so they must be handled with some care. Corms on the other hand are similar to flower bulbs. They can be left out of the ground for up to 2 weeks and they need no one to water or tend them, and can be boxed together for shipment.

In general, bananas are shipped out to the markets when they are still partially green, yet a banana is considered ripe and ready for eating when it is fully yellow and spotted with small brown spots. Occasionally, bananas will bypass the ripening room, and show up at the market still fully green; these nearly never ripen into quality fruit, if they ripen at all. The flavor and texture of many kinds of bananas are usually affected by the optimum temperature at which they ripen. Bananas spoil and turn grey at low temperatures and are only refrigerated down to 13.5°C during shipping.

2.5 Cultivated Banana Varieties in Malaysia

Banana cultivars can be differentiated based on their genomic groups or their ploidy level like AA, AAA, AB, AAB and ABB based on the morphological scoring method (Stover and Simmonds, 1987). Cultivars with the B genotype have starchy and acidic fruits and they are generally eaten boiled, fried or roasted. On the other hand, cultivars with the A genome have sweet and fine textured fruits, and they are mostly eaten raw or serve as a dessert. The popular dessert cultivars of Malaysia are Pisang Mas (AA), Berangan (AAA) and Rastali (AAB) whereas the popular cooking types are Pisang Raja (AAB), Nangka (AAB), Awak (ABB), Nipah (BBB) and Kapas (AAB) (Jamaluddin, 1990). Only Pisang Mas is grown to be exported outside and the others are for domestic consumptions.

Pisang Mas (AA) is the most important banana cultivar in Malaysia since they have high potential in exporting. The plant has a small fruit, ranging between 8.0-12.0 cm in length and 3.0-4.0 cm in diameter and the plant height is about 2.2-2.6 m. The peel is thin and golden yellow in colour once it ripe. The pulp is firm, yellow in colour, sweet-smelling and very sugary taste (Jamaluddin, 1990).

Figure 2.5.1: Pisang Mas. Source: Valmayor et al. (2000).

Pisang Berangan (AA) is next to the earlier cultivar in importance, a potential export crop. The plant is somewhat tall plant which is 2.5-3.0 m in height. The fruit is medium to large in size and the peel is thick, golden yellow in colour and enclosed with slight to heavy blemishes. The flesh of Berangan is very aromatic, dry and sweet (Jamaluddin, 1990).

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Figure 2.5.2: Pisang Berangan. Source: http://www.bananas.org/ (2009).

Pisang Raja Udang (AAA) can be in green mutation form and the ripen fruit of the red variety, It is extremely creamy to taste and not overly sweet. Bad thing is that the shelf life is very short upon ripening. The fruit is one kind of dessert type (Jamaluddin, 1990).


Figure 2.5.3: Pisang Raja udang. Source: http://www.bananas.org/ (2009).

Pisang Rastali (AAB) is an extremely popular dessert cultivar in Malaysia. The plant is a medium-sized plant of about 2.5 to 3.0 m in height. The fruit is small to medium and has a thin peel, yellow orange in colour when ripe and covered with moderate to heavy black blemishes and slightly sour in taste (Jamaluddin, 1990). The fruit detaches easily from the hand when ripe and should be eaten at the correct ripe stage, or else, the fruit is unappealing.


Figure 2.5.4: Pisang Rastali. Source: http://www.bananas.org/ (2009).

Pisang Raja (AAB) is an important cooking cultivar in Malaysia. The fruit is angular and the skin is thick and develops an orange-yellow colour when ripe. The flesh of Raja is creamy orange, very sweet and coarse in texture (Jamaluddin, 1990).

Figure 2.5.5: Pisang Raja. Source: Valmayor et al. (2000).

Pisang Nangka (AAB) is the cooking cultivar that is widely distributed throughout Peninsular Malaysia. They become a popular choice for farmers since they are vigorous plant with a high yield as well as early shooting. The plant is medium, 'heavy' and the fruit is long, pointed and angular (Jamaluddin, 1990). The peel is thick and remains green when ripe. The pulp of Nangka is creamy, fine texture, starchy and a subacid in taste. Have a distinct aroma when cooked.


Figure 2.5.6: Pisang Nangka. Source: http://www.bananas.org/ (2009).

Pisang Tanduk (AAB) is a cultivar that has largest fruit. They have restricted commercial value in Malaysia due to the poor yield. The fruit is huge, between 25.0-35.0 cm in length and 5.0-6.5 cm in diameter. The skin is yellow when ripe and the pulp is light creamy orange in colour, fine in texture but firm. The fruit also has tremendous keeping quality and remains starchy even when gully ripe. When cooked, the flesh will turn out to be very sweet in taste.

Figure 2.5.7: Pisang Tanduk. Source: Valmayor et al. (2000).

Pisang Awak (ABB) is a common in the northern dry states of Malaysia. The fruit is small to medium and turns yellow when ripe. The skin of Awak is thick and the pulp is whitish, firm as well as sticky (Jamaluddin, 1990). Creamy and not overly sweet fruit.


Figure 2.5.8: Pisang Awak. Source: http://www.bananas.org/ (2009).

Pisang Nipah (BBB) is widely grown in the southern states of Malaysia. They have a short, plump and angular fruit with a thick skin that becomes yellow when ripe (Jamaluddin, 1990). The pulp is creamy white, fine texture with a developed core. Even though the flesh becomes sweet upon ripening, the fruit is always cooked before consumption.


Figure 2.5.9: Pisang Nipah. Source: http://www.bananas.org/ (2009).

2.6 Molecular Phylogenetics

Molecular phylogenetics is a taxonomy method that used a short standard sequence of organismal DNA to infer it evolution relationship (Nei and Kumar, 2000). Before the advent of molecular biology technique, the evolution relationship was inferred based on morphologies only. How different they are from each other or any characteristics formed because of mutation or adaptation to the environment.

Early attempt at the molecular phylogenetic was using the protein, enzyme, carbohydrate and molecules which were separated using technique such as nbmkf;bchnique made the DNA sequence data widely available and accumulates in staggering speed. This accumulation of data is a major advantage to the evolution biologist because the DNA sequence itself is a document of evolution history (Felsenstein, 1985). In recent times, chemotaxonomy was largely replaced by the DNA sequence as the marker for the phylogenetics study.

Essential key for the molecular phylogenetics is in closely related organism; commonly have high degree of connection in the molecular structure of the organisms. Distantly related organisms show some degree of similarities. Conserved sequence such as mitochondrial DNA are expected to accumulate mutation over time, assumedly mutation occur in constant rate fashion. This constant rate of mutation provides a molecular clock for the evolution.

2.7 Chloroplast DNA

Comparative analysis of chloroplast genomes have verified extremely useful to make clear the evolutionary relationships and in phylogenetic study in angiosperms (Birky, 2001). Commonly, chloroplast DNA (cpDNA) is maternally inherited, of angiosperm species. The form of inheritance of cpDNA is a serious determinant of its molecular evolution and of its population genetic configuration (McCauley et al., 2007).

Chloroplast DNA is broadly used by plant evolutionary biologists for a multiplicity of reasons, including as a marker of seed movement in studies of population arrangement and phylogeography (Ennos, 1994; McCauley, 1995), and as a tool in studies of plant systematic (Olmstead and Palmer, 1994). A lot of these applications suppose maternal inheritance as a chief determinate of both the magnitude of gene flow and the mode of molecular evolution of the genome. Further, the tendency for angiosperm chloroplast genomes to be maternally inherited has led to the idea that they be considered as useful sites for insertion of engineered genes in genetically manipulated species due to the lack of transmission through pollen would deeply reduce the probability of ''escape'' (Gressel, 1999).

2.8 Genetic Markers

Genetic markers stand for the genetic differences among organisms or species. Commonly, they do not signify the target genes themselves but just perform as 'signs'. Genetic markers that are positioned in close proximity to genes perhaps will be referred as gene 'tags'. Those kinds of markers themselves do not have an effect on the phenotype of the trait of interest since they are located simply near or 'linked' to genes controlling the trait. All genetic markers reside in particular genomic positions inside the chromosomes known as 'loci'. There are three main types of genetic markers: (1) morphological or known as visible markers which themselves are phenotypic traits or characters; (2) biochemical markers, which include allelic variants of enzymes; the isozymes; and (3) DNA or molecular markers, which expose sites of variation in DNA (Jones et al., 1997; Winter and Kahl, 1995).

Morphological markers are typically visually characterized phenotypic characters such as flower colour, seed shape or growth habit. According on (Winter and Kahl, 1995), isozyme markers are differences in enzymes that are detected by electrophoresis and specific staining. The major disadvantages of morphological and biochemical markers are that they may be limited in number and are influenced by environmental factors or the developmental stage of the plant. Nevertheless, despite all those limitations, morphological and biochemical markers have been awfully functional to 171 plant breeders (Eagles et al., 2001; Weeden et al., 1994).

DNA markers are the mainly used type of marker primarily because of their abundance. They come up from dissimilar kinds of DNA mutations like substitution mutations, rearrangements (insertions or deletions) or errors in replication of tandemly repeated DNA (Paterson, 1996). These markers are selectively neutral since they are typically located in non-coding regions of DNA. Contrasting with the morphological and biochemical markers, DNA markers are basically limitless in number and are not exaggerated by environmental factors or the developmental stage of the plant (Winter and Kahl, 1995). DNA markers have various applications in plant breeding such as identifying the level of genetic diversity within germplasm as well as cultivar identity, instead of in the construction of linkage maps only (Jahufer et al., 2003; Winter and Kahl, 1995).

DNA markers can be classified into three classes based on the manner of their detection: (1) hybridization-based; (2) polymerase chain reaction (PCR)-based and (3) DNA sequence-based (Joshi et al., 1999; Winter & Kahl, 1995). DNA markers possibly will reveal genetic varieties that can be seen by using gel electrophoresis and staining with chemicals (ethidium bromide or silver) method or detection with radioactive or colorimetric probes. They are particularly useful if they can expose differences between individuals of the same or different species. These kinds of markers are called polymorphic markers, whereas markers that do not distinguish between genotypes are called monomorphic markers. Polymorphic markers could be described as codominant or dominant as well. This explanation is based on whether markers can discriminate between homozygotes and heterozygotes.

2.9 rpoC Gene

In recent times, a number of chloroplast DNA sequences with major homology to bacterial RNA polymerase subunit genes have been discovered and reported (Ohyama et al., 1986; Shinozaki et al., 1986; Sijben-Muller et al., 1986). Sijben-Muller et al. was the first who reported the sequences of a gene with homology to the gene for the α subunit of Escherichia coli RNA polymerase (denoted rpoA). This was followed by the publication of the complete chloroplast DNA sequences of Marchantia (Ohyama et al., 1986) and tobacco (Shinozaki et al., 1986) which contained a α-like subunit gene as well. Furthermore, these plants hold two additional chloroplast genes, perhaps restricted within a single transcription unit, with homology to the p' (rpoC gene product) and p (rpoB gene product) subunits of E. coli RNA polymerase. RpoC gene also synonymous with RNA polymerase, beta prime subunit.