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Section: Biology
Is it Easy to Be the Creator?

Is it Easy to Be the Creator?

An unprecedented rise in technology at the end of the Second Millennium was felt far and wide across science. As far as biology is concerned, the rise was due to the development of two new fields: genetics and its ‘scion’, namely molecular genetics, which allowed manipulation with DNA molecules as the carriers of genetic information beyond the living cell. Genetic engineers and biotechnologists came to the scene.

Armed with techniques for genetic and cell/tissue culturing, they learned to develop ‘new’ organisms and thus undertook the role of evolutionary process facilitator, if not the Creator himself.

Blue roses and golden rice…

The advancements in genetic engineering of plants found practical applications soon. The first transgenic plants were developed early in the 1980s, and just ten years later China boasted growing herbicide-resistant transgenic tobacco on an industrial scale. Today we witness a large-scale production of new cultivars developed from genetically modified plants totaling about 200 species!

The use of genetic engineering techniques for transferring genes, responsible for traits of commercial value, opened up major new opportunities for improving main crops. Transgenic plants resistant to herbicides, pests, viruses, and diseases have been developed, as well as the plants with a well-balanced amino acid content and a modified fatty acid content; ornamental plants with modified flower coloration like blue carnations, roses, and chrysanthemums.

We can cite the striking examples of monellin and thaumatin. These amazing proteins identified in the African trees Dioscoreophyllum cumminisii and Thaumatococcus danielli are a thousand times sweeter than sugar. Transferred into other plant species (for example, strawberry or apple), the genes controlling the synthesis of these proteins endow the fruits with incredible sweetness. Since sweetness is due to a protein, not a carbohydrate, such plants represent an excellent solution for diabetic and obese individuals.

One of the major advancements in genetic engineering is a new rice cultivar developed under the guidance of Professor Ingo Potrykus (Switzerland) and featuring increased levels of provitamin A, iron, and folic acid. Eating the ‘golden rice’ helps compensate for vitamin A deficiencies, which is especially important for the countries where this deficiency is marked. Researchers from Cornell University developed another high-yield rice cultivar with resistance to draught and low response to soil salinization. They transferred two Escherichia coli genes controlling the synthesis of the carbohydrate trehalose, which makes the rice plants resistant to high and low temperatures, into the rice genome.

Transgenic food and ornamental plants are already well known. Furthermore, interest in transgenic plants as biological factories producing new materials, biologically active substances and drugs is growing. They could become a new source of proteins for medical purposes, cheaper and safer than the traditional systems, which rely on bacteria, yeasts, insect and animal cell cultures. The development of transgenic ‘edible vaccines’, which effectively improve the performance of the animal and human immune system, holds a lot of promise.

The potential scope of use of transgenic plants is extremely wide. For example, plants contributing to environmental pollution control, including those targeting heavy metals and biopolymers, have now been developed. In research and development laboratories throughout the world new ambitious projects are underway.

Transgenes are coming!

Let us assess the share of genetically modified plants in the world’s total of the main crops and let the figures speak for themselves.

It is obvious that transgenic plants occupy ever increasing agricultural areas. Over eight years (1996—2003) the area, on which such plants are grown, has increased 40-fold worldwide, topping 67 million ha. By 2003, as many as seven million farmers in 18 countries have been growing transgenic plants.

Genetically modified plants are grown for commercial purposes in the Republic of South Africa, Australia, India, Romania, Uruguay, Spain, Mexico, the Philippines, Colombia, Bulgaria, Honduras, Germany, and Indonesia. The largest producers are the USA, Argentina, Canada, Brazil, and China. Incidentally, Russia is not on the list!

Seven developed and eleven developing countries grow transgenic plants, the share of the latter being one-third of the area (20 million ha). Crops resistant to herbicides (soybean, maize, rape, and cotton) and pests are most popular.

These data suggest that the results of genetic engineering of plants are in great demand and they are widely applied in agriculture. Nevertheless, genetic engineering and its means are still a sort of ‘black box’ for the public, which causes little trust in genetically modified products. This concern, however, is totally based on unawareness and ignorance. Let us consider the process and techniques used for obtaining transgenic plants and compare this trend with the traditional methods of plant selection.

Fantastic reality

What is genetic engineering all about? Essentially, this is a targeted experimental modification of an organismal genome by introducing separate new genes. A question arises: what is so special about these new ways that they have come to rival the long existing techniques of plant selection, which until recently underlaid the development of new plant cultivars?

New, required genes are introduced to the host genome in both cases; the difference is in the ways of delivery, and this difference is important.

In terms of the traditional methods of plant selection the required genes are pooled in a hybrid plant genome through pollination between the donor plants that carry the required traits, while in genetic engineering, the required genes are isolated (cloned) and purposefully transferred into recipient organism’s cells. Thus, the ways look different, but essentially they remain identical.

However, there is one inherent difference in the results. The traditional techniques allow only the genetic information from closely related crossing species to be pooled. Genetic engineering gives a unique opportunity to rearrange a plant genome using genetic information from completely different systems: viruses, bacteria, insects, and animals. Obviously, this makes the genome much more modifiable within the plant kingdom and removes natural barriers between taxonomically distant entities, for example, monocotyledonous and dicotyledonous plants.

Thus, the genetic engineering techniques appear to represent a very logical supplement to the traditional ones based on selection and genetics. They really can launch morphogenesis so far as no plant selection scientist would dare think of.

Genetic engineering against potato lovers

Bt-protected potato — the first transgenic plant in Russian selection

Potato is one of the most popular crops across Russia. It is therefore important for national food safety that this product be affordable and shipped on a regular basis. However, potato yields remain low, and farmers always deal with problem of protecting this plant from pests and disease.
The main ‘illegal’ potato eater is the Colorado beetle (Leptinotarsa decemlineata), which had conquered about 2.5 million ha of plantations by 1992. Estimated potential losses of yields due to the Colorado beetle amount to 4.1 million tons (or about 700 million US dollars). Since potato prevails in the private sector (over 90 %), where crops may absolutely fail unless adequately pretreated, some argue that the true losses are 2—3 billion dollars annually!
Traditional plant breeding practices are of little help here. Hence the urgent need for new cultivars developed on the basis of modern biotechnology; in particular, genetic engineering. Not only will new cultivars reduce losses of yields, but also pesticide loads on plantations, which will be for the benefit of human health.
The generally recognized strategy for developing GM plants with resistance to pests is the use of natural insecticides: the proteins (delta-endotoxins) whose genes were cloned from the Bacillus thuringiensis. Bt-protected maize, cotton, and potato were first implemented in agriculture in the USA in 1995—1996. Large-scale tests and long experience of using them in agricultural practices in a variety of countries confirmed that the foods made on the basis of Bt-cultures are safe for human health and the environment.
Based on these prerequisites, Russian scientists have started one of Russia’s largest projects aimed at bringing the achievements of biotechnology into crops - the development of Bt-protected potato on the basis of Russian potato breeds. This project will be descibed in one of the coming issues.
Dmitry Dorokhov, Candidate of Biological Sciences, Deputy Director of the Center of Bioengineering of the Russian Academy of Sciences

Modern techniques for transgenic plant development comprise several main stages:

a) cloning of required genes and development of genetic constructs that can be expressed (function) in plant genomes;

b) transferring the genetic constructs into the plant genome;

c) assessing the sustainability of the expression of the transferred genes, and choosing individuals for further finer selection work among transformed plants;

d) biosafety testing.

The progress in genetic engineering techniques allows genes to be cloned depending on the aims and goals of a particular biotech project. What are the separate stages of the cloning process? Consider potato.

Although in Russia high-yield potato cultivars adapted to a wide range of environmental conditions are grown in large amounts, about one-third of the potato harvest is lost due to disease and pests. The Colorado beetle, potato’s sworn enemy, invaded Russia about 50 years ago. Initially chemicals were used for Colorado beetle control, which were not safe for human health and the environment. A variety of biological techniques have since been developed as a safer alternative; one of which relies on the use of a specific protein synthesized by entomopathogenic bacteria Bacillus thuringiensis.

Upon reaching the insect’s intestines, the protein splits producing an activated toxin, which causes insect death. In some countries, an insecticide drug based on this protein has for years been produced in industrial settings in required amounts using various B. thuringiensis strains. The drug is sprayed onto the leaves and reaches the pest organism with the food. Naturally, spray droplets contaminate the environment; worse, thus applied, drug dosage can not be controlled.

What does genetic engineering have in store for preserving potato from this devastating pest? The solution is much the same with a little yet important difference: it is the plant itself which should produce the required protein in its leaves. To make it happen, the B. thuringiensis CryA gene is transferred into the potato DNA and becomes a part of the potato genome. Moreover, it is incumbent on the transferred gene to spring to action only in pre-selected parts of the plant, for example, in foliage, which we do not eat, and not in tubers, which we do.

DNA circles going round…

How is the genetic construct for transferring the isolated gene into the potato plant developed?

First, the insects (Colorado beetles) that died naturally over the bacterial entomotoxin are collected. The bacteria on insect bodies are collected onto special Petri dishes and allowed to grow colonies on nutrition media. The colonies are then selected under a microscope and DNA is isolated.

In bacteria, genes are located not only in the chromosomes (as in higher organisms), but also in plasmids, which are small circular DNA molecules. In particular, the required gene — CryA — is located on one of such plasmids. The isolated plasmids are processed using special enzymes, a sort of ‘nuclear scissors’, which cut plasmid DNA into separate fragments.

The fragment containing the required gene is transferred into the Escherichia coli plasmid, which serves as a ‘copy machine’, because it enables obtaining of the required gene in many copies. The genes are then transferred into the plasmid of a different bacterium species, Agrobacterium tumefaciens. This one deserves a special mention.

Agrobacteria, a “ferryman” for genes

The most popular technique for transferring alien genes into plant cells (especially when the plants are dicotyledonous) relies on the use of soil bacteria of the genus Agrobacterium, preferably A. tumefaciens, which produces crown galls on the plants.

Only two out of 14 Agrobacterium species identified to date can infect plants. By inserting fragments of their DNA into plant genome, they cause tumors to grow. These tumors, otherwise called galls, provide shelter and food for these bacteria.

The crown galls appear where the plant surface, lesioned or damaged by rodents, is exposed to agrobacteria. The agrobacteria do not infiltrate plant cells, but “dispatch” there a small DNA fragment on the plasmid between two signal regions. Essentially, these regions are repeats 25 base pairs in length, which are ‘recognized’ by special enzymes at the very instant the agrobacteria sticks to the lesioned plant cell. The enzyme cleaves the DNA strand between the two signal regions. Then various protective proteins transfer the DNA strand into the cytoplasm and eventually the plant cell nucleus. The bacterial DNA fragment inserts immediately into the plant DNA, and the bacterial genes start functioning as the plant’s own genes. As a result, the metabolic pathways of the plant change, and the plant cell begins synthesizing nutrients for agrobacteria.

This DNA fragment of the A. tumefaciens plasmid was named tDNA, where t stands for transfer. The tDNA played an outstanding role in developing the strategies for plant modification by genetic engineering: based on that unique feature of the bacteria, the genetic engineers replace the bacterial genes by the genes that are of interest. Good as this way may seem for alien gene transfer, it is not at all versatile, for it better suits dicotyledonous plants. It is of little use for monocotyledonous plants — a large group, which includes such important crops as cereals — since they are resistant to agrobacteria. In these cases, direct gene transfer techniques are employed, one of which provides the delivery of DNA fragments on gold or tungsten particles using a ‘genetic gun’.

It’s been a hard day’s night…

As soon as the DNA fragment has reached the plant cell and has been integrated into the nuclear genome, the cell acquires a new status: it is genetically modified. At the next stage, the experimenter should separate transgenic cells from non-transgenic ones, have them multiplied, and then retrieve full-fledged plants.

For that purpose, the gene responsible for resistance to the antibiotic that is baleful to the plant is also transferred into plants together with the required gene (CryA in the case of potato). The presence of the former gene in the plant cell allows it to survive and grow on special media with admixtures of the antibiotic, and the experimenter can see which cells are transgenic. For example, the transfer of the nptII gene makes plant cells resistant to the antibiotic kanamycin.

After separating the cells on the selective medium and after the selected transgenic cells propagate, the experimenter apply them to a special media with phytohormones for retrieving a whole plant. The regenerated plants are grown in vitro (in the glass tube) and then are taken to a greenhouse.

This is not the end, but the work comes to its new tedious phase. First, the presence of the transferred DNA fragment with the required gene

in the genome of these plants should be demonstrated. Then, it is necessary to prove that the transferred genes can exist and function in a new genetic environment. Furthermore, they should not interfere with the functioning of the other genes, that is they should not cause mutation, but persist and be passed to the offspring…

Russia as a transgenic offshore entity

So we have got well-functioning transgenic plants. But before they are put to practice, a thorough work on sanitary and hygienic examination awaits to determine the chemical content in the original and transgenic plants, to ensure that the biological value and assimilability of the foods prepared from genetically modified plants did not deteriorate; special studies are needed to guarantee that any component of the transgenic plants is not allergic, carcinogenic, toxic or mutagenic or has no impact on the reproductive function of animals and man. It is expected that the transgenic potato will be much safer than natural potato grown on a plot near a highway and generously stuffed with ‘good’ old insecticides.

Biosafety tests include risk assessment of further transfer of the transferred genes into other organisms, in particular into closely related species through natural pollination. Furthermore, the effects the new genes may have on the susceptibility of transgenic plants to pests and disease are to be studied, as well as the effects the genetically modified plants themselves may have on soil microflora and other components of the biocenosis.

In the countries, where transgenic plants are cropped, special commissions have been established to test and certify genetically modified organisms. In Russia, their certification is coordinated by the Interdepartmental Commission for Genetic Engineering Issues established by the Russian Government in 1997.

On the whole, biotechnological activity is regulated by more than 150 acts, resolutions, and standards, though there is neither such thing as an area under genetically modified crops in Russia, nor a Russian made transgenic cultivar. This is not due to the fact that Russia’s people said NO to genetically modified organisms, but this rather reflects the attitudes of the Russian government and society towards genetics, which in advanced countries is considered as one of the first priorities.

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