Scientists are using genetic engineering, method for creating new forms of plants faster than by traditional selection, techniques without modifying the main agronomic characteristics of basic strains.

In molecular selection the desired characters are obtained by insertion of individual sense genes into the plant genome. For example, plants resistant to diseases caused by phytopathogens (bacteria and fungi) are thus evolved. Another trend is in improving the taste qualities. These two problems can be solved simultaneously now and then.

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Tertiary structure of the thaumatin protein molecule in two projections: A) frontal; B) lateral.

Numerous proteins protect plants from diseases caused by phytopathogens (bacteria and fungi). Biologists have classified these proteins: by now about 10 groups are known, for example, the family of pathogenesis-related proteins (PR-5). It unites substances initially isolated from the West African thaumatococcus (Thaumatococcus danielli) and the common maize (Zea mays) and called by the Latin names of these plants: thaumatin-zeamatin-like substances. These substances were found to be widespread and in this or that form, present in virtually all plants, accounting for their resistance to phytopathogenic fungi, together with chitinase (chitin-destroying enzyme; chitin is present in the fungal mycelium).

Thaumatin is formed in the thaumatococcus by the time of fruit maturation. Spontaneous mutation of the gene coding for this protein must have developed in the course of evolution. As a result of this mutation the peripheral part of the protein molecule was modified, primarily with respect to the spatial location of lysine residues. Thaumatococcus fruit became very sweet and tasty for chimpanzees and gorillas. These primates started consuming this fruit and spreading its seeds, thus contributing to the evolutionary fixation of its character. It is noteworthy that only higher primates can perceive the sweet taste of thaumatin, due to specific features of their gustatory receptors. Neither insects nor lower animals feel it.

Today insufficient sweetness of fruits and berries is a serious problem for agricultural manufacturers in many countries-Spain, USA, Chile and Australia. Geneticists are trying to solve it. For example, scientists of our artificial Climate Station used the gene coding for the supersweet protein thaumatin-II (one of thaumatin-like proteins) in experiments aimed at improving strawberry resistance to phytopathogens. This protein is about 3,000 times as sweet as sucrose (by weight) and 100,000 times as sweet by molecular ratio. This naturally brought us to the idea: what if we use this gene not only for plant protection from disease, but also for improving the taste of fruits and berries?

Thaumatin-11 gene was cloned at Leyden University in the Netherlands in the early 1980s. Attempts at using this protein as a culinary and pharmaceutical sweetening agent, primarily for diabetics, were made. The recombinant gene (about 1,000 nucleotide bases) was introduced into the bacterial genome and the desired expression was attained: formation of a sweet protein. However, an attempt at its wide-scale production failed, as the problem of effective expression of an eukaryotic gene (that of a higher plant) in the genome of a prokaryote (organisms like bacteria having no discrete nucleus to the cell and the chromosome system) was not solved. None the less, a sweetening agent derived from natural thaumatin is manufactured now under trademark "thaline".

In the mid-1990s Unelever, an English-Dutch Firm, donated to Russian biologists; the DNA of the gene coding for thaumatin-II; based on this DNA, a vector*

* Vector in molecular genetics is a DNA molecule capable of incorporating foreign DNA and transferring it into cells whose hereditary characteristics are to be modified. Vectors are most often made on the base of plasmids. - Auth.

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Schematic structure of the binary vector.

for plant transformation was designed at our Station. The resultant combination, though very simple, proved rather effective: owing to the 35S viral promotor (initiating genetic information transmission) and the intronfree (having no noninformative sites) sense part with minimum signal sequences (regulating protein transport and location in the cell). Using this combination, scientists of many research centers of the Russian Academy of Sciences and the Russian Academy of Agricultural Sciences designed transgenic plants - thaumatin-II - producing apple- and pear-trees, as well as tomatoes, carrots, and potatoes.

Within a year our scientists tested several strains of the transgenic carrot, including field trials (in collaboration with Institute of Selection and Vegetable Culture Seed Growing, Russian Academy of Agricultural Sciences). The studies showed that these plants were resistant to two pathogenic fungi species, but the root crop was not sufficiently sweet. Tomatoes obtained through the introduction of the thaumatin-like protein gene exhibited both increased resistance to phytofluorosis when grown on protected soil and marked sweetness of the fruit, too. Polish scientists achieved similar results for tomatoes and for cucumbers with an analogous insertion. Using the same construction, our colleagues at the Bioengineering Center of the Russian Academy of Sciences (Moscow) obtained a transgenic potato highly resistant to many phytopathogens.

We attained the best results in experiments with garden strawberry (Fragaria ananassa). A four-year cycle of field trials of its transgenic strains was carried out, and forms with improved taste were selected. On the other hand, these strains are better resistant to gray mold (Botrytis cinerea), a disease to which none of the known strawberry strains is immune and causing the greatest loss in crops.

To obtain a transgenic strawberry the gene coding for thaumatin-II was transferred into its genome by agrobacterial transformation. The Ti-plasmid is used for this purpose; this plasmid is a small annular molecule carrying hereditary information of an agrobacteri-um and responsible for the growth of tumors in plants infected with it. The Ti-plasmid contains a T-DNA site, capable of being incorporated into the plant genome. The assigned genes are inserted into this very site. In our case a binary vector was designed, with the thaumatin-II gene at the site of the bacterial plasmid T-DNA. In addition, the antibiotic resistance gene was inserted into the vector, and thus it became possible to identify and select the transformed cell from among ordinary cells devoid of such resistance (kind of labeling).

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Transgenic strawberries.

Regeneration of adventive sprouts from transformed strawberry cells.

The resultant vector was transferred into the agrobacterium. Strawberry leaves, pre-cultured in vitro under sterile conditions in artificial nutrient media at present temperature and illumination, served as explants (material from which a whole plant is then regenerated). Plant tissue was infected with agrobacteria carrying the plasmid with the thaumatin-II gene at a temperature of about 23°C in darkness. Then cells with the functioning insertion of foreign genes and growing in a medium in the presence of antibiotics were selected for several months.

Next came regeneration of strawberry plants from solitary transformed cells, which is rather difficult for cultured sorts, in contrast to model plants (tobacco or arabidopsis), and which often involves negative side-effects (somatic mutations and variations, chromosome aberrations, and other events leading to quality losses).

Multiplying the newly created strawberry plants, we obtained more than 20 strains carrying new hereditary information. Further analysis, however, showed that the thaumatin-like protein gene was not manifest in all of them. Some transgenic strains did not exhibit the expected immunity to the gray mold agent in trials of resistance to this agent even under laboratory conditions. In a geneticist's lingo this means abortive thaumatin-II gene expression: the gene is "silent". This phenomenon is often caused by random incorporation of the gene in the chromosome and its possible location in the zone where it exhibits by poorly the characteristics it encodes. The gene can be damaged during transfer, even "prolapse" or be modified (e.g. methylated). Another probability is when the marker (antibiotic resistance gene in our case) is transferred, while the sense gene is not.

In addition to transgenic strawberry resistance to phytopathogens, we controlled the taste of berries, their size, and plant morphology. All these characteristics cannot be evaluated under laboratory conditions or in a hothouse; field trials are needed. It is a mandatory stage of analysis of transgenic plants before their practical use. It is well-known that in classical selection of plants (crossing of two forms) just two or three hybrids are selected from among ten thousand. The use of gene engineering methods, e.g. regeneration from a single cell, can lead to various modifications even not related to gene transfer, as a result of which the characteristics of the new plant will differ from the initial strain.

Field trials were carried out on the test field of the Institute of Fruit Culture Selection, Russian Academy of Agricultural Sciences, in the city of Orel (the test field was certified by Inter-Departmental Committee for Gene Engineering). Transgenic and non-transgenic strawberry was planted in the same field sections, sever-

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Field trials of transgenic plants.

al plants of each strain being set at random (randomized), so as to level out the impact of soil and other factors on their growth. Soil and agrochemical control was carried out simultaneously. During flowering the plants were isolated by film caps in order to prevent uncontrolled pollen transfer.

At the end of the season only 3 strains were selected from among 24. These strains were characterized by all the desired qualities: presence of the sense gene (coding for the thaumatin-like protein), presence of the protein, and good production characteristics, since the final goal was to obtain transgenic strawberry plants without morphological changes, such as stunted forms. Their taste had to be better than that of the parental strain, and they had to be resistant to the main pathogen (gray mold). But most of the lines had flaws: the phenotype either differed from the initial strain or the taste did not improve even in the presence of the gene. Only five lines produced sweeter berries, as proved statistically.

Now we are working to improve the gene engineering design in order to increase the level of sense gene expression and achieve targeted location of the protein in the cell. For this purpose we insert the thaumatin gene with various signal sequences and analyze its cell location. We have upgraded the construction used for the first time in 1996 for obtaining transgenic strawberry.

We provided more accurate targeting of gene expression products and get them to accumulate in various cell structures. Protein location in plant tissues proved to be very important for the effective manifestation of the desired characteristic. For example, if protection from phytopathogens is our aim, the protein should be located in the cell-to-cell (intercellular) space. If the aim is taste improvement, the protein should be accumulated in vacuoles.

It is possible to do without antibiotic resistance genes when creating transgenic cultures, which is important for the subsequent industrial cultivation of these cultures, particularly for those consumed fresh. In such cases genes coding for assimilation of mannose (instead of sucrose) as a source of carbohydrates are used. This method was called "positive selection", in contrast to "negative" one, using antibiotic or herbicide resistance for cell labeling.

By now we have improved the resistance of transgenic strawberry to the gray mold agent by 25 - 30 percent. It is a very good result, given that absolute immunity is impossible.

In 2002 the scientists of our station patented the method for growing transgenic plants highly resistant to phytopathogens. In 2005, with support of the Foundation of the Russian Academy of Sciences for patenting Russian inventions, we received the international patent on our method for obtaining apple-trees as well as tomato and strawberry plants with improved taste characteristics and high resistance to phytopathogens.

The studies described in this paper have been in progress for 10 years. The Ministry of Agriculture of the Russian Federation is showing great interest in our laboratory's work in designing transgenic cultures of domestic strains and in securing the biological safety of their cultivation. It's a fact that apples grown in Central Russia often taste sour. So we are working to make them sweeter.

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