The genus of Nicotiana tabacum is member of the family Solanaceae that contains 75 species. Some of important species are: Nicotiana rustica, Nicotiana clevelandi, Nicotiana otta and Nicotiana tabacum. Nicotiana tabacum is the well-known species which spreads all around the world. Table 2.1 described the detail taxonomy of Nicotiana tabacum and Figure 2.1 shows the Nicotiana tabacum with enlarge flower (Ivan A. Ross, 2008; Vinod kumar, 1988).
Table 2.1: Taxonomy of Nicotiana tabacum
Taxonomy of Nicotiana tabacum
TracheobiontaÂ -- vascular plants
MagnoliophytaÂ -- angiospermes, angiosperms, flowering plants,
MagnoliopsidaÂ -- dicots, dicotylédones, dicotyledons
SolanaceaeÂ -- nightshades, solanacées
NicotianaÂ L. - tobacco
TabacumÂ L. -- cultivated tobacco
Nicotiana tabacum has been used widely as medicinal herb, crop plant and trade commodity in many different cultures and cultivated by human being for thousands of years. In the past, the species of Nicotiana tabacum was utilized, as a model plant in genetic-engineering research and plant-cell cultures. For higher plants, Nicotiana tabacums are applied as a model organism because of high homogeneity and high growth speed, featuring behavior of plant cell. Due to N. Tabacum's economic value and importance as a biological research tool, it has been focused to study its origin, organization and genome structure. (Ivan A. Ross, 2008; Amran, 2000; Watson, 1979)
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Figure 2.1: Nicotiana Tabacum plant with 4 times enlarge flower Scalebar=4cm (left) Scalebar=1cm (right)
The flowers of Nicotiana tabacum have terminal or sub terminal position in the form a raceme as a cyme. In addition, the terminal has several flowered inflorescences, the tube 5-6 cm long and 5 mm in diameters, widened in the lower calyx and upper throat, lobes generally triangular, white-pinkish with pale violet or carmine colored tips tube yellowish white (Feyza, 2004).
N. tabacum is one of amphidiploids which is thought to be created from two progenitors. In addition, Kenton et al. (1993), described on cytological evidence, have what a portion of the N. tabacum genome originated from N. sylvestris and an introgressed hybrid between N. tomentosoformis and N. otophora (Nita Sachan, 2004).
Nicotiana tabacum contains many alkaloids and the major alkaloid is nicotine. Nicotine's biosynthesis is managed by many kind of factors such as, phythormones, developmental age and biotic and abiotic stress. Nicotine and related alkaloid play a key function in plant defense against herbivore and insect attack in nature. In plants, the nicotine and related alkaloids initiates with the putrescine and/or polyamine, formed by one of two pathways. In the roots of N. tabacum, the nicotine biosynthesis happens exclusively. After synthesis, nicotine and other N. tabacum' alkaloids are shifted from the roots through the xylem to the leaves, in that they are collected in high density (Nita Sachan, 2004).
The soil and climate conditions influence N. tabacum under growth and alkaloid formation . In addition, it is sensitive for ground humidity, air, type of soil and physical property. The best temperature is from 20 to 30 Â°C for growth; and atmospheric humidity of 80 to 85%. N. tabacum is a local of tropical and subtropical America, and generally, the plant seeds are sown in the spring (either outside or in a greenhouse), the plants blows in summer and the tobacco is ripe for harvesting at the beginning of autumn. But in the last century, N. tabacum is commercially cultivated in all tropical countries (Wikipedia, Watson 1979).
2.2 The Culture Environment and Media Component
Basic requirement are shown in Table 2.2. For growth and development, the growth medium has to provide all essential mineral ions required. Many plant tissue cultures as plant growing they will carry out photosynthesis and also required the addition of a fixed carbon source in the form of a sugar. Physical factor, such as pH, the gaseous environment, light and temperature, also have to be maintained within acceptable limits.
Table 2.2: Some of the elements important for plant nutrient and their physiological function. These elements have to supply by the culture medium in order to support the growth of healthy cultures in vitro
Component of proteins, nucleic acids and some coenzymes
Always on Time
Marked to Standard
Element required in greatest amount
Regulates osmotic potential, principal inorganic cation
Cell wall synthesis, membrane function, cell signaling
Enzyme cofactor, component of chlorophyll
Component of nucleic acids, energy transfer, component of
intermediates in respiration and photosynthesis
Component of some amino acids (methionine, cysteine)
Required for photosynthesis
Electron transfer as a component of cytochromes
Component of some vitamins
Enzyme cofactor, electron-transfer reactions
Enzyme cofactor, chlorophyll biosynthesis
Enzyme cofactor, component of nitrate reductase
The compositions of the medium, mainly plant hormones and the nitrogen source have profound effects on the tissues that grow from the initial explants. For growth and development, the growth medium must be provide with all essential mineral ions required, and it has to be provided with additional organic element such as amino acids, vitamins (Adrinal et. al., 2003; Hortman and Kester, 1983).
In conclusion the plant tissue culture has three basic components:
An organic supplement supplying vitamins and amino acid;
A source of fixed carbon; usually supplied as the sugar sucrose.
The essential elements are separated into the following groups:
Macronutrients or Macroelements,
Micronutrients or Microelement;
An iron source.
Combined several different components makes plant cell culture as appendix 1 (Feyza 2007; Adrinal et al., 2003 )
2.5 Plant Growth Hormones
A phytohormone can be defined as a small that elicits a physiological response at very low concentration. Plant growth hormones are critical media components in determining the development pathway of the plant cells.
In this study, the project used two types of plants hormones such as auxin and cytokinin for the plant's growth. The addition, cytokinin and auxin are necessary for callus growth.
.Phytohormones mainly are divided into the following categories (Figure 2):
Figure 2.2: Structures of representatives of five classic plant regulators
The growth regulator requirements for most callus cultures are auxin and Cytokinin. Two class of plant growth regulator (phytohormone) will be analyzed.
The hormone promotes both cell division and cell growth. The most important member Auxins family is indole-3-acetic acid (IAA). On the other hand IAA applies in plant culture media is limited. IAA is unstable too both heat and light ( Davis (2004). Other Auxins are available (table 2.3).
Table 2.3: Commonly used auxins, their abbreviation and chemical name
Miller and Skoog (1957) noted that a high amount of cytokinin and low amount of auxin forms shoots. A high amount of auxin and a low amount of cytokinin promotes roots in the media.
The most generally discovered natural auxin is IAA. O n the other hand, depending on the species, season, age of plant, and conditions under which it has been growing.
Cytokinins are a group of phytohormone substance active in promoting cell division, differentiation and some other physiological processes. They are most complex class of plant growth substances, and the hormone has two form; natural and synthetic. The hormones are also thermostable. Cytokinins are divided into following categories (see table 2.4) ( Adrinal et.al., 2003; Roberta, 1992; Loisus, 1982).
Table 2.4: Commonly used cytokinins, their abbreviation and chemical name
2iP (IPA) b
a Synthetic analogues.
b Naturally occurring cytokinins.
2.6 The Current Study of Nicotiana Tabacum
"Tobacco plants can also be easily regenerated from tobacco leaf pieces. Leaves are cut into aproximately 1 cm squares with a sterile scalpel (avoiding large leaf veins and any damaged areas). The leaf pieces are then transferred (right side up) to gelled MS medium supplemented with 1mg l-1 BAP (a cytokinin) and 0.1mgl-1 NAA (an auxin). Over the next few weeks, callus forms on the explants, particularly around the cut surfaces. After 3 to 5 weeks shoots emerge directly from the explants or from callus derived from the explants. When these shoots are about 1cm long they can be cut at the base and placed on to solid MS medium without any plant growth regulators. The shoots will form roots and form plantlets that will grow in this medium and can subsequently be transferred to soil" (Adrinal et.al., 2003).
2.7 Nicotiana tabacum and biotechnology
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Nicotiana tabacum has been applied as a model crop in the development of fresh techniques, in recent researches. For model researches on gene transformation, N. tabacum is a brilliant matter. For diverse purposes, genes have been moved to plant, with advances in gene technology. For the improvement of N. tabacum species, many helpful of genes have been transferred into the plant genome to produce new traits. A number useful of genes are related to resistance to insect, herbicide and tolerance to environmental stresses.
Consequently, in recently years, many kinds of transgenic Nicotiana tabacum have been produced with important traits. The gene encoding for the endotoxin of Bacillus thuringiensis (BT) had been introduced in transgenic N. tabacum plants. And new transgenic plant demonstrated to be efficient in controlling lepidopteron insect's pests. Studies on the gene transformation of N. tabacum have been made to obtain a higher expression level in order to increase the effectiveness of this technology. The most important insecticidal movement was determined in the transgenic N. tabacum. In plants, alterations to the bacterial gene sequence of BT endotoxin were created for more readily expressible and were efficient to obtain resistance against less sensitive pests.
The budworm Heliothis armigera feeds on at least 120 cultivated plants, such as Nicotiana tabacum. Larvae infest the leaves or buds of the plant. And the larvae of insects cause damage with big hole. (Louis, 1982; Tran 2006).