SU1708849A1 - Method of plant genetic transformation - Google Patents

Abstract

The invention relates to genetics, biotechnology and can be used in the cultivation of all cultivated plants. The purpose of the invention is to increase the efficiency of transformation. The method consists in the fact that certain genes are transferred to the plant genome by means of a Ti plasmid based vector. containing a marker gene (antibiotic resistance), the pollination being carried out after the application of plasmid DNA in a solution of sucrose, boric acid and Ca (NO) 2, and the selection of transformants is carried out on selective media, in particular the Knop environment. containing kanamycin. The method allows for the quick and efficient generation and selection of transformants. The frequency of transformation is 0.6-1.45%.

Description

The invention relates to genetics, biotechnology and can be used in the selection of cultivated plants.

There is a known method of genetic transformation of plants, which includes carrying out a L4 microinjection of whole DNA into plant protoplasts.

There is also known a method according to which exogenous DNA is introduced into plant protoplasts by the method of electroporation.

However, these methods are not very suitable for many monocotyledonous plants, in particular, cereals. The application of these methods is difficult. that for cereals it is usually difficult to get a plant from protoplasts.

There is also known a method of genetic transformation involving the use of Agrobacterium bacteria.

tumefaclens for gene transfer using vectors based on the T1 plasmid.

This method is practically unacceptable for monocotyledonous plants, since A. tumefaciens does not have virulence to them.

There is a method of genetic transformation of plants, for example, tobacco, which consists in that. that the pollen is germinated on medium containing exogenous DNA in the form of a T1 plasmid. Sprouted pollen is applied to the stigma of the pistil. Tissue cells are then analyzed for nopaline synthetase activity, on the basis of which a genetic transformation is judged.

There is also known a method of genetic transformation of plants, according to which the stigmas of plants are incubated in the presence of plasmid DNA. containing antibiotic resistance gene, in particular to

kanamitiyu. Plasmid DNA was introduced into the pollen grains of plants using an electroporation method, then the plant was pollinated and the selection of transformants in the resulting progeny was performed for kanamycin resistance.

However, the known methods are unacceptable for plants in which the viability of pollen largely depends on the storage time, for example, maize and other cereals.

The closest to the proposed method is that pollen is collected from maize plants, a paste is prepared from a mixture: pollen, solution sucrose, exogenous DNA, exogenous DNA is isolated from maize lines containing the marker genes of epidermis staining n The prepared mixture is applied to the stigmas of the plant pistils. The frequency of transformation is estimated by the frequency of occurrence of colored grains on the cobs.

The disadvantages of this method are that

transformation is non-vectorial;

no easily selectable mar-. Kern ui gene, the presence of which would quickly and efficiently select transformants;

there is no possibility of obtaining a sufficient number of seeds necessary for analysis, due to their weak entrainment.

The purpose of the invention is to increase the efficiency of transformation.

The method involves treating the pistil stigma with a mixture of pollen and exogenous DNA in a sucrose solution, using a T-plasmid vector, for example, the PGV series, containing the antibiotic resistance marker gene, npvi, this plasmid is mixed with sucrose solution, boric acid and Ca (MOH) 2, the mixture being immediately applied to the stigma of the pistil and immediately pollinated, and the selection of transformed plants was carried out by germination on a Knop medium, g / l: Ca (Li) 2 0, S; KH2P04 0.2; MgO-i x 7H20 0.2; KNOa 0.2; FeP04 0.1, containing kanamycin at a concentration of 250-400 μg / ml.

Mixtures of the following composition are prepared: 0.40, 5 M sucrose; 0,017-0,020% boric acid; 0.038-0.042% Ca (N03) 2; PH 5.5-6.8; 40-100 μg / ml plasmid DNA (for example, PGV1501) containing the kanamycin resistance gene.

It is 6 p 1. In a mixture of 0.4 M sucrose, 0.018% boric acid, 0.04% Ca (MO3) 2, (pH 5.5-5.8), immediately before applying to the stigma of the pestles, enter t plasmid DNA (PGV 1501) containing the kanamycin resistance gene in an amount of 70 μg / ml. On every ear of corn

Rfs lines (previously isolated) were immediately applied with 200 µl of the mixture, and then immediately pollinated with its own pollen. After ripening, seeds are germinated in a Knop medium. 1-2-day old seedlings are then transferred to Knop medium containing kanamycin at a concentration of 400 µg / ml. After 10-14 days. the plants that have been selected on this medium are transferred from the soil under the conditions of an air-conditioner (20 lux - 16 h, temperature

the day is 28 ± 1 ° C, nights 19 ± 1 ° C) and grow for transplantation into open ground.

Some of the plants at the seedling stage are analyzed by Southern blot hybridization to prove integration into

plant genome of the gene of interest.

The frequency of transformants obtained using the proposed method is about 0.6%.

Example 2. In a mixture containing 0.45

M sucrose, 0.017% boric acid, 0.038% Ca (NO3) 2, pH 5.5-5.8, are plasmid DNA containing the kanamycin resistance gene. The mixture is applied to the stigma of the tomato pistil and immediately pollinated. Ripe seeds are germinated in a Knop medium with kanamycin (100 μg / ml). When using a tomato variety Torch, the transformation frequency is 1.45%.

In the prototype for one gene frequency

transformation is about 0.3%, i.e. obtained, as a result of using the proposed method, the frequency of transformation is 2 times higher than that obtained in the known method. Analysis of transformanto. By the Sauern blot hybridization method showed that the kanamycin resistance gene was inserted into the plant genome,

Thus, the evaluation of the next generations of putative transformants by selection on selective media and by blot hybridization method shows the effectiveness of this transformation method.

The method allows for the quick and efficient generation and selection of transformants. The method also makes it possible to reduce the time between the collection of pollen, the preparation of the mixture and the pollination of plants, which does not cause a noticeable effect of physical factors and nucleases on DNA, pollen and pollen tubes (mechanical ruptures, degradation, etc.). The method can be used for the targeted transformation of the plant genome.

Claims (1)

Invention Formula A method of genetic transformation of plants, including mixing exogenous DNA with plant pollen, applying the mixture to the pistil stigma in sucrose solution, selecting transgenic plants, characterized in that, in order to increase the efficiency of transformation, a vector based on a T1 plasmid carrying antibiotic resistance and an inserted gene. Pollen blending is carried out in a strain of 0.4-0.5 M sucrose, 0.017-0.020% boric acid, 0.038-0.042% Ca (MO3) 2, pH 5.5-5.8, after applying to the stigma of the pistil, immediately pollination, and the selection of transgenic plants is carried out by germination on a Knot medium containing kanamycin in a concentration of 250-400 µg / m. 0