RU2351121C1 - Method of obtaining transgenic monocotyledon plants - Google Patents

Abstract

FIELD: genetic engineering.

SUBSTANCE: invention can be used in monocotyledon plants selection for creation of novel sorts and hybrids by means of genetic engineering, in works insertional mutagenesis, separating and cloning of plant genes. In order to obtain transgenic monocotyledon plants in period of their active blooming flowers lacking own fertile pollen are selected. As object of genetic transformation, blooming female gametophyte is used, which is processed with suspension of strain Agrobacterium tumefaciens with activated vir-genes, pistil filaments being processed directly. After that said flowers are pollinated with pollen of fertile plants. For processing cells with strain Agrobacterium pistil filament sections, located near flower ovary, are used.

EFFECT: said operations allow to create transgenic monocotyledon plants preserving high frequency of their obtaining under conditions, that correspond to natural temperatures of blooming, and to simplify technology of obtaining transgenic plants.

4 cl, 1 dwg, 1 tbl

Description

The invention relates to agriculture and biotechnology and can be used in the selection of monocotyledonous plants when creating new varieties and hybrids using genetic engineering, in the work of insertional mutagenesis, isolation and cloning of plant genes.

A known method for producing transgenic corn plants based on the agrobacterial transformation of immature maize embryos in an in vitro culture (Zhao Z.-Y., T. Cai, Tagliani L. et al. Agrobacterium-mediated sorghum transformation // Plant Mol. Biol., 2000, v.44, pp.789-798), according to which immature maize embryos are placed for 5 min in a suspension of agrobacterial cells (density 0.5-1.0 × 10 ⁹ cells / ml) in an inoculation medium containing mineral salts and vitamins according to MS , casein hydrolyzate (1.0 g / l), 2,4-D (1.5 mg / l), glucose (36 g / l), sucrose (68.5 g / l), acetosyringone (100 μM), after which they are transferred to 3 days on co-cultivation redundant, which differs from the initial 2,4-D concentration increased to 2.0 mg / L, sucrose and glucose concentrations reduced to 20 and 10 g / L, respectively, the addition of L-proline (0.7 g / L), ascorbic acid (10 mg / l), MES (0.5 g / l) and agar (8 g / l). Then the embryos are transferred for 4 days to a medium without acetosyringone, but with the addition of the antibiotic carbenicillin (100 mg / l) to inhibit the growth of agrobacteria. Then, to induce embryogenic callus, the embryos are transferred to a medium that differs from the previous one by the absence of glucose and a reduced concentration of 2,4-D (up to 1.5 mg / L) and the addition of the selective agent phosphinotricin (5 mg / L), the resistance gene to which (bar) was present in the T-DNA of the used agrobacterial strain, and cultured for 2 weeks. The surviving (stable) callus is transferred to a medium of the same composition with an increased level of phosphinotricin (10 mg / l) and transplanted every 2 weeks. To develop embryoids, callus is transplanted onto a medium that differs from the previous kinetin addition (0.5 mg / L), and then onto a shoot formation medium that differs from the previous one by the absence of 2,4-D, an sucrose concentration increased to 60 g / L, replacing kinetin with zeatin (0.5 mg / l), with the addition of indolyl-3-acetic acid (IAA) (1 mg / l), abscisic acid (ABA) (0.1 µM) and thadiazuron (0.1 mg / l). The shoots are transferred to the rooting medium - 1/2 Murashige-Skoog (MS), (nitrile-3-acetic acid (NAA) (0.5 mg / l), indole-3-benzene acid (IBA) (0.5 mg / l), sucrose (20 g / l), agar (7 g / l), and then on medium of the same composition, but without IAA and IBC. The plants obtained using molecular genetics methods analyze the presence of a fragment of foreign DNA in their genome (T -DNA of agrobacteria). This method allows to achieve a stable transformation frequency of up to 10.1% based on the number of inoculated embryos.

The disadvantage of this method is the high complexity and duration of the process of obtaining transgenic plants. In addition, the use of an in vitro culture system, firstly, can lead to somaclonal variants among transforming plants, and secondly, limits the use of this method, since not all maize samples (selection lines and hybrids) give a well-growing embryogenic callus with high regenerative ability, or their explants producing embryogenic callus necrotize as a result of co-cultivation with agrobacterial cells.

A known method for producing transgenic monocotyledonous plants using genetic markers used in genetic engineering in higher plants to select transgenic plants, for which the nptII gene is used, which encodes the neomycin phosphotransferase enzyme, which determines resistance to kanamycin. For the selection of transgenic plants carrying the nptII gene from the seeds collected from transforming plants, the embryos are extracted and grown on a nutrient medium with kanamycin (Danilova S.A. Optimization of the agrobacterial method of transformation of corn. Abstract. Diss. Cand. Biol. Sciences. M., 2001). Moreover, seedlings carrying and expressing the nptII gene have a green phenotype, while seedlings that do not carry this gene become albino and die.

This method, which allows one to select kanamycin-resistant transformants with the nptII gene from certain species of dicotyledonous and monocotyledonous plants, has, however, two significant drawbacks: the high complexity, limiting the volume of the analyzed material, the duration of contact of the embryos with kanamycin during cultivation on a kanamycin-containing medium As a result, the development of even kanamycin-resistant transformants is disrupted.

Closest to the proposed method is a method for producing transgenic sorghum plants (Patent for the invention of the Russian Federation No. 2229793, IPC: A01H 1/00, C12N 15/82, C12N 15/00), including the transformation of sorghum cells using Agrobacterium tumefaciens and selection of transgenic plants. To obtain transgenic plants, flowering sorghum panicles lacking their own fertile pollen are used, which are pollinated with pollen of fertile sorghum plants and then a suspension of cells of the Agrobacterium tumefaciens strain with activated vir genes is applied, and acetosyringone is used to activate vir genes, and the suspension of Agrobacter cells tumefaciens is carried out at a temperature optimal for the transformation process, while transgenic plants are selected among seedlings grown from seeds tied to these panicles.

The disadvantage of this method is the requirement to carry out transformation at a temperature optimal for the transformation process, which contradicts the natural temperature optimum for pollen maturation, since it is believed that the optimum temperature for activation of vir genes is below 25 ° С (Fullner KJ, Nester EW Temperature affects the T-DNA transfer machinery of Agrobacterium tumefaciens // J. Bacteriol. 1996. V.178. P.1498-1504), and the optimum temperature for pollen maturation is a temperature above 25 ° C. To avoid this contradiction, plants have to be processed at night, grown in special vessels (or dig plants from the ground and placed in vessels) and transferred for incubation at low temperatures in special rooms. After incubation at low temperatures, reverse movement of the plants is required until complete ripening under conditions of optimal illumination and temperature, which greatly complicates the technology for producing transgenic plants, leads to plant losses, impaired ripening and, ultimately, to a decrease in the efficiency of obtaining transgenic plants.

The objective of the invention is to provide a faster and easier way to obtain transgenic monocotyledonous plants while maintaining a high frequency of their production under conditions corresponding to the natural flowering temperatures of monocotyledonous plants.

The technical result is the ability to carry out highly efficient DNA transfer from agrobacteria to plants at real natural flowering temperatures of monocotyledonous plants (above 25 ° C), with a simplification of the technology for producing transgenic plants (processing, incubation, ripening procedures).

This object is achieved in that in a method for producing transgenic monocotyledonous plants, including selecting as an object of genetic transformation flowers of monocotyledonous plants deprived of their own fertile pollen, pollinating flowers with pollen of fertile plants, treating flowers with a suspension of Agrobacterium tumefaciens cells with activated vir genes, according to the proposed solution as an object of genetic transformation, a flowering female gametophyte of a monocotyledonous plant is used, which is first treated with a suspension of cells it is a strain of Agrobacterium tumefaciens with activated vir genes and then pollinated with pollen from fertile plants.

In addition, pollinating of a flowering female gametophyte with pollen of fertile plants is carried out within an hour after they are treated with a suspension of cells of the strain Agrobacterium tumefaciens, and the suspension of cells of a strain of Agrobacterium tumefaciens is treated directly with pistil filaments of a flowering female gametophyte, while the flowers are selected during their active flowering. For treatment with a suspension of cells of the Agrobacterium tumefaciens strain, sections of pistil filaments of a flowering female gametophyte located near the ovary of a flower are used. To obtain transgenic maize plants for treatment with a suspension of cells of the Agrobacterium tumefaciens strain, sections of pistil filaments of flowering female gametophyte located at the outlet of the cob are used.

The invention is illustrated by a drawing (photograph), which shows evidence of the incorporation of T-DNA into the plant genome in the form of DNA fragments of a certain length obtained as a result of polymerase chain reaction (PCR) and visualized by agarose gel electrophoresis, in particular, the PCR analysis of DNA from seedlings of corn transformed in planta, where the numbers 1-8 indicate the paths: 1 - DNA from an unformed plant (negative control); 2 - molecular weight marker (Fermentas, Latvia), fragment size starting from the top: 3000, 2000, 1500, 1200, 1031, 900, 800, 700, 600, 500 bp; 3-8 — test seedlings whose grains germinated without delay on kanamycin.

The proposed method is as follows. Female flowers (as part of monoecious or dioecious, dioecious) plants are isolated with parchment insulators before flowering. To process the flowers, a suspension of Agrobacterium tumefaciens cells with a cell density of 1 × 10 ⁷ cells / ml, to which acetosyringon is added, is used. Female flowers of a plant after treatment with an agrobacterial suspension are pollinated with pollen from the same or another line of the same plant species and again isolated. Seeds are collected from treated plants, germinated and seedlings carrying the marker gene are selected on kanamycin medium. A tissue fragment is taken from kanamycin-resistant seedlings and the presence of the DNA sequence of the transferred gene is checked using PCR (polymerase chain reaction) using untreated and surface-sterilized plants. Seedlings carrying the marker gene DNA fragment ("PCR +" seedlings), which are transgenic plants, are selected.

The inventive method was implemented on corn plants of the AT-3 line and sorghum. Plants of the same line served as pollen donors. Transgenosis was performed using Agrobacterium tumefaciens, using strain GV3101, with plasmid pTd33, in which there was a selective marker gene nptII, which determined resistance to kanamycin. The cell suspension of these agrobacterial strains was grown on a rocking chair on CIB nutrient medium (Fullner KJ, Stephenens KM, Nester EW An essential virulence protein of Agrobacterium tumefaciens, VirB4, requires an intact mononucleotide binding domain to function in transfer of T-DNA. // Mol. Gen. Genet. 1994. V.245. P.704-715) to a density of 1x10 ⁷ cells / ml at room temperature. Upon reaching the required density, acetosyringone (200 μM) was added to the suspension and either directly used in the transgenesis experiment or placed in a refrigerator (t ° = 4-7 ° C) for use over the next 2-3 days.

Before flowering, the cobs were isolated with parchment insulators. As an object of genetic transformation, a flowering female gametophyte of a monocotyledonous plant was used, the pistillate strands of which were located at the exit of the cob threads were cut off and first treated with a suspension of Agrobacterium tumefaciens strain cells with activated vir genes, and then pollinated by the pollen of fertile plants for an hour was pollinated by shaking from a bag or fingers on the surface of a cut bundle of pollen tubes. The temperature during the processing of plants varied depending on the year in the range from 20 to 28 ° C. To identify transgenic plants, seeds collected from cobs treated with an agrobacterial suspension and untreated (control) cobs of the plants of the initial line were sterilized with a 5% chloramine solution (15 min), washed intensively in running water, soaked in distilled water (24 hours), re-sterilized in a laminar box with a 5% solution of chloramine and without washing was laid out on an agar medium containing kanamycin (200 mg / l), and after the appearance of roots, it was transferred to the soil in vessels with soil after 2-5 days, where it was germinated within 2-3 weeks.

Confirmation of the transgenic nature of the obtained green seedlings was carried out using the PCR method (see drawing). In this case, DNA was isolated from the leaves of selected plants according to generally accepted methods (Maniatis T., Frisch., Sambrook J. Molecular cloning. Laboratory manual. M .: Mir, 1984). The reaction was carried out with oligonucleotide primers specific for the 250 bp nptII gene fragment: ACAGACAATCGGCTGCTCTGATG and GGCAGGAGCAAGGTGAGATGACA. The reaction products were separated by agarose gel electrophoresis; phage λ DNA was used as a molecular weight marker. The plasmid pTd33 was used as a positive control.

The result of DNA analysis showed that in a number of studied samples, the total DNA of the plant contains a sequence of about 500 bp, characteristic of the nptII gene. Based on the number of seedlings grown, the frequency of adult transformed plants obtained from them was 1.3–6.8% (see table 1). Moreover, it was noted that the frequency of transformation does not depend on the increased ambient temperature (more than 25 ° C).

Table 1. The frequency of insertion of T-DNA into the genome of the corn after processing the pistillate strands of corn A. tumefaciens according to the proposed method Options Temperature during plant treatment 25-28 ° C 20-22 ° C Seed sampling 1200 600 The number of green seedlings on kanamycin 53 37 The number of seedlings analyzed by PCR 53 37 The number of PCR-positive seedlings (% of the total number of seedlings) 25 (47%) 9 (24.3%) The number of transformants (% of the total number of seeds) 6.8% 1.5%

Thus, the above data show that the inventive method allows to obtain transgenic plants with high frequency at real natural flowering temperatures of monocotyledonous plants more than 25 ° C with a significant reduction in complexity and simplification of the technology for producing transgenic plants (processing, incubation, ripening).

The proposed method allows: 1) to obtain transgenic plants at real natural flowering temperatures of monocotyledonous plants while simplifying and increasing the speed of processing technology, incubation, growing plants while maintaining a high transformation frequency, 2) to obtain transgenic plants bypassing the in vitro culture system, which accelerates and simplifies the procedure for obtaining transgenic corn plants, 3) to avoid the occurrence of somaclonal variation; 4) to avoid the chimericity of transformants resulting from their development from multicellular meristems containing both transformed and non-transformed cells; 5) use for transformation almost all known varieties and lines of corn, because there are no restrictions on genotypes capable of forming a well-growing embryogenic callus.

Claims (4)

1. A method for producing transgenic monocotyledonous plants, including selection of flowers of monocotyledonous plants deprived of their own fertile pollen as an object of genetic transformation, pollination of flowers with pollen of fertile plants, treatment of flowers with a suspension of Agrobacterium tumefaciens cells with activated vir genes, characterized in that the genetic object transformations use a flowering female gametophyte of a monocotyledonous plant, which is first treated with a suspension of cells of the Agrobacterium tumefaciens strain with activated vir genes And then pollinated with pollen fertile plants. 2. The method according to claim 1, characterized in that the pollination of a flowering female gametophyte with pollen from fertile plants is carried out within an hour after their treatment with a suspension of cells of the strain Agrobacterium tumefacien. 3. The method according to claim 1, characterized in that the treatment with a suspension of cells of a strain of Agrobacterium tumefaciens is carried out directly by the pistillate strands of a flowering female gametophyte, while the flowers are selected during their active flowering. 4. The method according to claim 3, characterized in that for the treatment with a suspension of cells of a strain of Agrobacterium tumefaciens, sections of pistil filaments of a flowering female gametophyte located near the ovary of a flower are used.

Images