Gene Transfer in Plant through Protoplast | Plant Tissue

In the below mentioned article, we will discuss about gene transfer in plant through protoplast.

Since the isolated protoplast shows high picnocytic activity, so it can uptake some spe­cific genes or foreign DNA and this property brings some genetic modification in plants.

Ge­netic modification by DNA uptake implies that DNA from one source be taken up and incorpo­rated into the recipient genome and that genetic information encoded in the exogenous DNA be expressed as a new and stable characteristic in the recipient protoplast and subsequently in the regenerated plants. This approach provides an alternative way to increase genetic diversity to the existing genetic make-up of the plant.

Several approaches has been employed for transferring exogenous DNA or specific genes into the protoplasts such as:

(a) Direct gene transfer.

(b) Indirect or liposome mediated delivery of DNA to plant protoplasts.

(c) Gene transfer using biological vectors.

(d) Microinjection of DNA into protoplasts.

(e) Fusion between the bacterial spheroplasts and plant protoplast.

Direct uptake of foreign DNA has been in­vestigated in protoplast. The anticipated steps in the uptake process into protoplasts include binding to specific sites on the plasma membrane and passage through the membrane and cyto­plasm. This is followed by binding to specific binding sites, passage through the nuclear mem­brane into the nucleus and ultimately integration into the host genome (Fig 12.9).

A major obstacle in feeding isolated DNA directly into the protoplasts is the presence of en­zymes that degrade DNA. So it needs a good pro­tection for exogenous DNA so that DNA makes a safe journey from external medium to the re­cipient nucleus and such protection will facilitate its stabilization in recipient cytoplasm.

The use of liposome is a new innovation to facilitate the uptake of nucleic acid without any degradation. Liposomes are the liquid crys­talline structure obtained when amphipathic lipids such as phospholipids are dispersed in wa­ter or aqueous salt solution. Each liposome is a bilayered completely enclosed sac-like vesicle (250At o 10 µm diameters).

It is possible to en­close the nucleic acid within liposome which can readily transport through biological membrane of the protoplast. The result indicates that lipo­some can protect the enclosed nucleic acid from the degradation by the enzymes of the recipient protoplast.

The incorporation of DNA by vectors may also reduce the degradation of DNA and improve the gene transfer process. Two types of vectors are being considered, plant viruses and bacterial plasmids. Using the DNA virus as a vector, it may be possible to insert the foreign DNA into the viral genome Since the foreign DNA in as­sociation with viral DNA can be incorporated safely in the nucleus of the recipient cell. So DNA viruses are more or less suitable vectors for gene transfer.

Plasmids are double stranded, closed circu­lar extra-chromosomal DNA found in bacteria. Plasmids can integrate into the chromosome of the recipient cells. The research with plasmids has developed rapidly in the recent years and has resulted new and revolutionary techniques for gene transfer. On the other hand, a series of novel enzymes called restriction endonucleases have been discovered.

Such enzymes could be used as ‘molecular scalpel’. The enzymes cut the DNA at points with specific nucleotide se­quences. At the same time ligase enzyme used as ‘molecular adhesive’ can join the cleaved DNA. The restriction enzymes cleave the plasmid DNA to linear, double stranded sections with overlap­ping and complimentary nucleotide sequences.

The linear plasmid DNA section can be mixed with endonuclease-cleaved section of foreign DNA from other source. With the help of ligase, reconstituting the plasmid DNA carrying the genes or section of foreign DNA can be mixed with the protoplasts. After the hybrid plasmid is taken up by the protoplast, it is replicated and the genetic information that is encoded in the foreign DNA can be transcribed and even­tually expressed in the walled cells and subse­quently in the plants derived from the proto­plasts (Fig 12.10).

It is well known that the pathogenic bacte­ria Agrobacterium tumefaciens, causes the plant tumour crown gall upon wounding and infection. The agents responsible for tumour induction are plasmid called tumour inducing or Ti plasmids. Crown gall cells have their capacity to grow in vitro in absence of phytohormones. It has been established that the autonomous growth of the tumour cells is caused by the integration of a piece of DNA (T-DNA) form a Ti plasmid.

This naturally transformed system or Ti plasmid DNA can be used in experiments to transform plant protoplasts into hormone independent opine synthesizing cells. Thus genetic manipu­lation of plant protoplast by means of Agro-bac­terium tumefaciens can be achieved by three dif­ferent approaches:

(a) Fusion of crown gall pro­toplasts with normal protoplast,

(b) Infection of cell wall regenerating protoplast with virulent strains of A. tumefaciens,

(c) Transformation of protoplasts with Ti plasmid DNA.

With the help of above genetic engineering or plasmid technology, intensive efforts are being made in various laboratories, all over the world to transfer the ‘nif’ gene (nitrogen fixing gene) in the protoplast of non-legumes. It may well usher us into an era of “self fertilized farming”.

Microinjection of foreign DNA into the nu­clei of protoplasts has also been developed into an efficient and reproducible method of gene transfer.

Recently, the fusion of protoplasts with bac­terial spheroplasts are also used as a method of introducing DNA into plant cell. Genetical change that occurs in bacteria due to absorption of foreign DNA or extract of other bacteria, is known as bacterial transforma­tion. Trans-genesis is a new term for the type of transformation in higher plant system.