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Abstract
INTRODUCTION
Plants have always played a major role in the treatment of human and animal diseases. Medicinal plants are a therapeutic resource much used by the traditional population of the world especially for the health care. World wide interest in the use of medicinal plants is now increasing. Beneficial effects of plant-based medicines and other plant-based products are being rediscovered (Batanouny, 1999).
One of the most important medicinal plants is Datura metel LINN (Thorn apple). It is an annual herb belongs to the family Solanaceae. The plant is about 1-1.5m high and has unpleasant odour. The flowers are white, the seeds are found in small fruit which are completely covered with short and sharp spines. Seeds are yellow to light brown and have an unpleasant taste (Kotb, 1983).
Datura plants have many arabic names such as Shejret el- Janna; Tatura and Nefir. The entire plant is used many years ago in traditional medicine. The whole plant is used for asthma and as a sedative. The leaf extract is an ingredient in remedies for cough and chest complains. Plant boiled in water is taken to ease asthmatic breathing. Dried leaves and flowers are smoked as tobacco to relieve difficult breathing. Seeds are taken as analgesic in small doses. The seeds of ripe fruits are burnt and smoke inhaled for treating epilepsy (Boulos, 1983). Datura plant is a very important medicinal plant as it is a known source of the tropane alkaloids; hyoscyamine, atropine and scopolamine (hyoscine). The total alkaloid yield has been estimated to be between 0.06 and 0.50%. The young leaves contain mainly scopolamine (hyoscine), whereas hyoscyamine is the major constituent of the mature leaves. Hyoscine (C17 H23 NO4) has anticholinergic, antiasthmatic and antispasmolitic effects. Hyoscyamine (C17 H23 NO3) has similar chemical structure to hyoscine, but it is smaller only by a single molecule of oxygen. Hyoscyamine has the same pharmaceutical effects as hyoscine. Atropine is the racemic from of (–) – hyoscyamine, which effects the nervous system. Atropine is used in eye drops preparation. It is also used to treat nerve gas poisoning, Parkinson’s disease, peptic ulcers, diarrhea and bronchial asthma. In addition to these alkaloids, the plant contains other minor tropane derivatives, as well as chlorogenic acid and lectins. Furthermore the seeds of Datura metel contain up to 30% fixed oil and about 0.2% alkaloids (Dewick, 1997).
Over the last two decades, plant cell cultures have proved to be a valuable tool for the study of the biosynthesis of secondary products such as tropane alkaloids in plants especially D. metel. Plant cell culture techniques have many advantages compared to conventional agriculture methods. It is easy to manipulate culture conditions, to feed precursors, and to extract the tissues for pharmaceutical compound production. Establishing dedifferentiated cultures from organized plant tissues were a major goal of early plant cell culture studies. Today, this is a relatively routine procedure which is an essential prerequisite for a range of subsequent approaches including regeneration, embryogenesis growth of large-scale cultures and selection strategies. The key to establishing callus and cell suspension cultures is the choice of the optimum culture medium component, proper explant source, and plant growth regulators concentration. Many novel plant culture techniques are now standard laboratory practice and several are already under full-scale industrial application. This proves not only the value of the methodology to science and to world agriculture, but also that it can be an economically viable alternative, extension to existing procedures (Roberta, 2000).
Technologies for introducing foreign genes into cells are critical for both basic studies of gene function and production of transgenic organisms that have commercial value. A diverse set of techniques have been developed for gene transfer to microbial, plant and animal cells. Some systems utilize biological vectors such as reteroviruses or Agrobacterium to accomplish gene transfer. Others rely on chemicals (e.g. calcium phosphate precipitation, polyethylene glycol or lipozymes) or physical means (i.e. microinjection or electroporation) to facilitate DNA transfer across cell membranes (Klein et al., 1992). Agrobacterium- mediated transformation method is commonly used to transfer foreign genes to plants. It is used successfully to transform several plant species such as tobacco, pea, carrot, celery, peanut, arabidopsis, and even other recalcitrant plant species such as rice and wheat. It is also used to transform some medicinal plants such Datura innoxia (Narula et al., 2004), Datura candida (Rhodes et al., 1994), Atropa belladonna and Hyoscyamus niger (Kanegae et al., 1994).
Active ingredient such as tropane alkaloids and their derivatives as any other biochemical compound used in preparing medicinal drugs may have many side effects. One of the most and important side effects is the rule of such extracted material on chromosomal structure and behaviour. This can be organized through cell division stages as chromosomal aberration.
The present study aimed at using plant cell culture and biotechnology techniques to throw light on the following points:-
1. Establishment of aseptic source material by germination of Datura metel seeds in lab.
2. Establishment of calli cultures from different explants.
3. Study the effect of growth regulators on callus and tropane alkaloids production.
4. Study the effect of different growth regulation on direct regeneration.
5. Establishment of Datura transformation protocol by using Agrobacterium tumefaciens carrying GUS gene.
6. Evaluation and determination of tropane alkaloids from the different obtained cell lines.
7. Study the effect of total tropane alkaloids at different concentration on the chromosomal behaviour of onion root tip cells.