Approaching to transgenic technologies
Asim Munawar1*, Aqsa Arshad1, Muhammad Ishaque Mastoi2, Shehbaz Sharif1 and Muhammad Ali3
1Department of Entomology, University of Agriculture Faisalabad.
2Department of Plant and Environmental Protection, NARC, Park Road Islamabad.
3 Department of Clinical Medicine and Surgery, University of Agriculture Faisalabad.
Agricultural researchers have long recognized a major drawback to breeding as a means of introducing valuable traits: It takes years, or even decades, to assemble all of the desirable traits in the same organism. This factor has driven the development of techniques that can bypass traditional breeding, by modifying genes directly in the genome of the plant or animal. There are two possible outcomes of direct gene modification: gain of function or loss of function. In gain-of-function experiments, a gene is added to the organism, or a gene’s expression or function is altered so that it performs a new function or expresses in a new pattern. This approach has been used extensively in plants for the introduction of genes that confer herbicide resistance or act as pesticides. Growth-hormone genes have been introduced into animals in order to increase their size and growth rate. This is the case for deleterious traits or, for example, genes that cause organ transplants from pigs to be recognized by the human immune system. The best way to remove a gene is by homologous recombination. Unfortunately, this technique has proven to be very inefficient in both plants and animals. Greater success has been achieved by using antisense or interfering RNA (RNAi). Detailed descriptions of these techniques are given in the chapter on high-throughput genetics. Both antisense and RNAi have been used successfully in both plants and animals to reduce levels of specific genes or their products.
- Transgenic plant technology:
Mechanism of gene inserting
One of the most widely used techniques for introducing genes into plants makes use of the soil bacterium Agrobacterium tumefaciens. When placed in contact with a wounded plant cell, Agrobacterium is able to transfer a portion (the T-DNA) of its Ti plasmid (shown in the upper image on the slide) into the genome of the plant. In the wild, the T-DNA carries genes that co-opt the plant’s metabolism forcing it to produce substances that feed the bacteria. The infected plants make large tumorous growths known as galls (shown in the lower image on the slide). In the early 1980s, Jeff Schell and Marc Van Montagu showed that the T-DNA could be modified to replace these genes with a useful gene. The modified T-DNA can then be returned to the Agrobacterium. The Agrobacterium containing the modified plasmid is allowed to grow on a plant surface that has been slightly wounded. The wounding causes a signal to be released that tells the bacteria to transfer the T-DNA into the plant genome. On the modified T-DNA is a gene that confers antibiotic resistance. When grown on the antibiotic, only plants that contain the modified T-DNA will survive.
- Transformation of rice:
Different steps during making transgenic
The first figure illustrating how Agrobacterium tumefaciens transfers the TDNA to a plant cell, where it becomes integrated into the plant’s genome. The second figure illustrates the various steps in transformation of plants. The top part of second figure shows rice callus tissue (undifferentiated cells) growing on media containing an antibiotic. The calli in the petri dish on the right have been transformed with T-DNA that confers antibiotic resistance. In the petri dish on the left are calli that were not transformed and are dying from the antibiotics. The T-DNA also contains a marker gene that produces an enzyme able to make a blue substance. The transgenic callus tissue on the top right of second figure turns blue when incubated with the substrate for the marker enzyme. In the middle panel of second figure antibiotic-resistant shoots are seen emerging from the callus. The shoots are allowed to grow into plantlets while on media and then are transferred to soil (lower left). A leaf turns blue when incubated with the substrate, showing that it contains the transgene. Seeds from transgenic plants are able to germinate on media containing antibiotics while the non-transgenic seeds in the petri dish beside them are not able to germinate.
- Transgenic animal technologies:
Transferring foreign gene into animal nucleus
Pioneered in mice in the early 1980s, microinjection with thin needles can be used to introduce genes directly into the nucleus of a fertilized embryo, as shown in the image. This technique has been used to produce transgenic pigs, cattle, and sheep with genes such as an enhanced version of the growth-hormone gene. A major problem with this technique is its low efficiency. Another problem is that the microinjected DNA inserts randomly in the genome. This condition can result either in aberrant expression when the DNA is inserted near a strong enhancer element or very low expression when the DNA is inserted into heterochromatin. Furthermore, it is difficult to control the number of copies of the inserted gene, frequently resulting in large tandem duplications.
- Gene knockout techniques:
Homologous recombination has been shown to work in animals and plants, but again, the efficiency is quite low. Gene targeting via homologous recombination in Arabidopsis. The Yanofsky lab at the University of California, San Diego, introduced a portion of the AGL5 gene involved in flower development that had a gene inserted into it which confers resistance to the antibiotic kanamycin. The researchers were able to select plants that were kanamycin resistant and whose genes had undergone homologous recombination. After homologous recombination, these plants were mutant for the AGL5 gene. Approaches using RNAi are just beginning to be used in farm animals to reduce expression of a gene. Of potentially great value is the ability to combine gene targeting of individual cells with nuclear transfer (described on the next slide) to generate animals with specific genotypes.
Genes targeting via homologous recombination (Plate#4)
The post-genomic era offers unrivalled opportunities for the complex genetic manipulation of plants towards useful ends. Our increasing understanding of metabolic pathways and identification of the genes involved provide the basic tools for producing hardier crops that could resist disease and thrive in adverse environmental conditions, whilst having enhanced nutritive value and health-promoting properties. Plant raw materials, such as fibres, oils and starch, could be improved to allow more cost-effective and environmentally benign processing by industry, and entirely new industrial and therapeutic products could be produced in crops in a sustainable manner. This information is necessary to understand the biology of transgenic.