BIOTECHNOLOGY

Plant cell and tissue culture
Many procedures in plant biotechnology depend on the ability to manipulate and grow plant cells in culture. Sometimes it is possible to generate new kinds of plants by such manipulations. Mutations in individual somatic cells would often go unnoticed. This somatic variation can be uncovered by culturing and regenerating new plants from the cells of a plant. This is called"somacloning". Cell walls can be stripped from cells with digestive enzymes to produce protoplasts. Protoplasts of different species can be mixed together and encouraged to fuse with special solutions (often containing polyethylene glycol). In this way"somatic hybrids" can be formed that might be difficult or impossible to get by conventional hybridization:

Brassica leaf protoplasts

Protoplasts are very delicate, but can often be cultured and will develop new cell walls. Then it may be possible to encourage embryo development (somatic embryogenesis) and the regeneration of new plants.

Genetic manipulation
Further development of biotechnology involves the ability to put new genes into plants, creating transgenic organisms (often called GMOs, genetically manipulated organisms). One procedure for this involves direct injection of DNA into a plant cell:

A much more common technique uses special strains of the crown gall bacterium, Agrobacterium tumefaciens. This organism injects a circular piece of DNA, or plasmid, into a host plant cell as part of its normal infection process. This plasmid can get integrated into the host DNA and then passed on through mitosis and meiosis to future generations.

The plasmid in wild-type Agrobacterium causes tumor development in plants. Gene transfer is accomplished using"disarmed vectors", plasmids that have been modified by deletion of the genes promoting tumor development. The circular DNA of the plasmid can be opened using restriction enzymes and a gene construct inserted to reclose the circle. Agrobacterium does not infect all plants. Another general method of gene transfer is particle bombardment: tiny metal particles are coated with DNA and fired into plant cells or tissue using a bullet or a blast of compressed air:

Whatever method is used genes are introduced only into a few cells and it is necessary to sort out the few transgenic cells from many that are unchanged. The most common way of doing this is to include a"reporter" gene for antibiotic resistance in the DNA construct. Also it is necessary to include a promoter so that the genes will be expressed in their new environment. A promoter from cauliflower mosaic virus is commonly used because it causes a high rate of transcription of mRNA from the introduced genes. The transformed cells or tissue will be cultured on a medium containing an antibiotic, such as kanamycin and only the cells transformed with the whole construct will survive. Because they express the gene for antibiotic resistance they are also likely to contain the other gene in the construct.

Finding genes
The techniques of genetic manipulation have become almost routine, but a major problem has been to know what genes to insert. Every eukaryotic cell contains many thousand genes and each is structurally complex:

In addition to the promoter, already mentioned, the DNA sequence usually contains non-coding regions, or introns among the coding sequences, or exons. Transcription starts  produces a primary mRNA transcript that is edited to remove the intron segments. A poly-adenine tail is added so that the mRNA will be recognized by a ribosome and used to direct protein synthesis. Translation will start at a special methionine codon (AGU) and ends at a stop codon (UAG). Because of the difficulties of working with genomic DNA researchers often look at the RNA produced by cells in order to find gene sequences. If a group of cells shows a particular phenotype, such as disease resistance, they must contain RNA coding for the protein causing the phenotype. It may be possible to isolate this RNA and use it to construct an artificial gene that can be put into another plant to make it resistant to the disease. DNA can be made from RNA using an enzyme from certain viruses called"reverse transcriptase". Usually it will be necessary to analyze the sequence of bases in the gene; then probes can be made from radioactive or fluorescent bases to match the sequence so that the gene can be detected as it is transferred from one organism to another:

complemetary sequence of probe binding to gene

Sections of DNA are often manipulated by cutting them with restriction enzymes. These are enzymes produced by certain bacteria that recognize and cut in particular base sequences in DNA:

Eco R1, an example of a restriction enzyme

If two different DNA molecules, such as a promoter and a gene are cut with the same restriction enzyme, they can be joined by DNA ligase to make a new construct:

The construct can be put into a plasmid vector in a similar fashion.

Genomics

Although it is difficult to work with genomic DNA, we can only understand how genes are regulated and work together by looking at the detailed structure of the chromosomes. That is why there is a major effort to decypher the complete DNA sequence of representative organisms from each of the kingdoms. The human genome project is expected to lead to medical advances. Arabidopsis thaliana (thale cress,a member of the Brassicaceae) was chosen for the plant genome project. Arabidopsis has and unusually small genome and is easy to grow in the laboratory. Many interesting mutants have been discovered and many of these have been located on particular chromosomes.

Copyright © Michael Knee,
The Ohio State University
All rights reserved.