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Protoplast cultivation

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Salma Samaha

on 3 October 2013

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Transcript of Protoplast cultivation

Protoplast Cultivation
Medicinal plants are the most important source of life saving drugs for the majority of the world’s population. The biotechnological tools are important to select, multiply and conserve the critical genotypes of medicinal plants.
Definition :
The entire plant cell without its cellulosic cell wall is known as plant protoplast. It has been described as naked plant cell because the cell wall has been removed either by a mechanical or an enzymatic method.
Protoplast culture is the aseptic isolation & culture of protoplast in vitro with the obtaining of viable plant.

Importance of protoplast culture
1. Two or more protoplasts can be induced to fuse to produce a hybrid plant.

2. After removal of cell wall the isolated protoplast is capable of ingesting foreign material .

3. For the studying of cell wall biosynthesis & deposition.

4. Population of protoplasts can be studied as a single cellular system that is their manipulation is similar to that of microorganisms.

Source tissue
Tissue for protoplast isolation consisted of :
1 - Fully expanded leaves excised from
greenhouse-grown plants
2 - Actively-growing shoot cultures
3 - Cotyledons of aseptically-germinated seedlings
4 - Placental tissue of fruits
5 - Friable embryogenic callus

Protoplast purification
1 – Removal of debris & enzymes

2 – Removal of broken protoplast

3 – Protoplast viability and cell wall formation test.
TomTato' tomato and potato plant unveiled in UK A plant that produces both tomatoes and potatoes, called the TomTato, has been developed for the UK market.

What is grafting?
• Grafting is the process of combining two different plants to create a single one
• It requires lots of skill and practice, but has been successfully achieved by providing a clean cut on the two plants and taping the ends together until they heal
• The purpose is to combine one plant's qualities of flowering or fruiting with the roots of another that offers vigour and resilience
• Most plants need to be grafted within their own genus - such as potatoes and tomatoes - but it is sometimes possible to graft those of a differing makeup

Factors affecting protoplast culture
1 – Plant species & varieties
2 – Plant age & organ
3 – Pre-culture condition
4 – Pre-treatment of tissue before isolation of protoplast

Methods of isolation
1. Mechanical method
In this method large & highly vacuolated cells of storage tissue such as onion bulbs, scales, radish root & beet root tissue could be used for isolation. The cells are plasmolysed in an osmotic solution, causing the protoplast to shrink away from the cell wall.

2. Enzymatic method
This method generates very large no. of protoplast in comparison to mechanical method.
First Isolated cells are prepared (maceration) by treating the leaf or other tissue segment with macro enzyme (Pectinase) in 13%mannitol. Pectinase mainly degrades the middle lamella while cellulose are required to digest the cell wall. The cells are purified by filtration through nylon mesh. Then the cells are incubated in 2% cellulose for about 90 min.

The aggregation of two or more protoplasts is not enough to start a fusion. Protoplast surfaces bear strong negative charges. In contrast to animal cells is the surface charge not caused by sialic acid residues but by phosphate groups. Intact protoplasts in suspension do thus repel each other. They can be very impressively linked and fused by the addition of calcium ions or polyethylenglycol. Electric fields are an alternative.

Sexual hybridization occurs when haploid cells generated in a previous meiosis fuse. The fusion of somatic diploid cells should generate a tetraploid fusion product provided that the nuclei fuse, too. If this is the case, it is spoken of a synkaryon. A fusion product where the nuclei stay separate is called a heterokaryon.
Fusion of Protoplasts & Hybridization
Done by :

- Ahmed Al - Maken
- Salma Samaha
- Fatima Ebrahim
- Mohammad Al-Hussaini
- Mohammad Abd Al-Naser
- Mohammad Naser
- Mahmoud Nassar
- Menna Al-Sayed
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