RU2420060C1 - Method of genetic transformation of plants of meadow clover selection-valuable samples - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: samples of morphogenetic tissue with sprouts of meadow clover are cut into parts with size of 3-5 mm, which are placed onto Gamborg medium B₅ with 2 mg/l of 6-benzylaminopurine. The upper surface of explant cut is inoculated with an agrobacterium. Co-cultivation is carried out within 48 hours, afterwards explants are washed from agrobacteria remains on Gamborg medium B₅ of the same composition with addition of 50 mg/l of kanamycin and 500 mg/l of cefotaxime. Regeneration of plants with roots is carried out on the medium of the same composition, but without cefotaxime, provided there are no signs of agrobacterial infection. Then, using PCR-analysis, availability of inbuilt genes is checked, and selection-valuable criteria preservation of initial samples is inspected.

EFFECT: new method of genetic transformation of plants of meadow clover selection-valuable samples is proposed.

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Description

The invention relates to the field of agriculture and can be used in plant breeding for the directed creation of the initial breeding material of meadow clover with economically valuable traits by the method of genetic transformation, as well as in studies of physiology, phytopathology and plant genetics.

There is a method of regeneration of meadow clover plants during genetic transformation [1], including cultivating a transformed morphogenic clover culture on a Hamburg B ₅ nutrient medium with the addition of kanamycin and cefotaxime, and the morphogenic culture is obtained without the formation of dedifferentiated tissue by culturing a hypocotyl on Hamburg B ₅ nutrient medium , 0 mg / l of 6-benzylaminopurine, 1.0 mg / l of kinetin and 0.05 mg / l of α-naphthylacetic acid with further transplantation of explants on fresh medium of the same composition with 2.0 mg / l of 6-benzylaminopurine, and the epicotyl part of the seedlings is maintained by culturing in Hamborg medium B ₅ without hormones or by micropropagation in a medium of the same composition with the addition of 2 mg / l of 6-benzylaminopurine. Morphogenic cultures in the process of genetic transformation retain the ability to regenerate plants and rhizogenesis up to the 24th passage or more on the Hamburg B ₅ nutrient medium with 50 mg / l kanamycin. Morphogenic cultures obtained from hypocotyl explants by genetic and morphological characteristics do not differ from the initial genotypes.

The disadvantages of the method include the fact that it is limited to describing the features of plant regeneration during genetic transformation.

There is information in the literature on the method of genetic transformation of meadow clover plants in an in vitro tissue culture [2], which involves inoculating the surface of explants placed on Hamburg B ₅ medium without antibiotics using a culture of agrobacteria, coculturing for 48 hours, and further cultivating ex- plantings on Hamburg B ₅ medium with 50 mg / l kanamycin in the presence of an antibiotic eliminating agrobacteria, plant regeneration. Transformed plants were regenerated from callus tissues formed from petioles of young leaflets of plants grown in a greenhouse.

The disadvantages of this method include the following: 1) in experiments, the regeneration of transformed plants was carried out from callus cultures, which significantly increases the risk of undesirable somaclonal variation and, as a result, a possible change in the selection traits of the starting material; 2) the use in experiments of explants from plants grown in a greenhouse, significantly increases the cost of maintaining an appropriate regime of cultivation during the year and the fight against pests and diseases; 3) the creation of recipient systems based on plants that have not passed preliminary selection for regenerative capacity (PC) unjustifiably increases the number of experimental options due to the need to bookmark parallel PC control options and subculture them throughout the entire process of genetic transformation.

The purpose of the invention is the development of a method of genetic transformation for directional changes in one or more target characteristics of clover plants of meadow selection-valuable samples.

In the proposed method, the goal is achieved in that the genetic transformation includes inoculation of the surface of explants placed on Hamburg B ₅ medium without antibiotics, culture of agrobacteria, cultivation for 48 hours, further cultivation of explants washed from agrobacteria on Hamburg B5 medium with 50 mg / l kanamycin in the presence of an antibiotic eliminating agrobacterium, plant regeneration and PCR analysis of the presence of integrated genes. Inoculation is carried out by applying an agrobacterium with a spatula to the cut surface of parts 3-5 mm in morphogenic tissue with shoots placed on Hamburg B ₅ medium with 2 mg / l of 6-benzylaminopurine, followed by in vitro regeneration of plants by direct regeneration, without the formation of callus tissue, and PCR analysis of DNA isolated from young aseptic leaves and petioles of in vitro regenerated plants, which formed roots in the presence of a selective factor of kanamycin at a concentration of 50 mg / l, and control the conservation of selection-valuable (acid stability) features of the original sample.

The method is as follows. Morphogenic cultures with small shoots having high PC were cut into 3-5 mm pieces, placed with a slice up on Hamburg B ₅ agar medium with 2 mg / L 6-benzylamnopurine (BAP) without cefotaxime and kanamycin, and inoculated with overnight culture of agrobacterial strains carrying various genetic constructs with marker (kanamycin resistance) and target genes. The inoculum was applied with a spatula only on the surface of the slices, excluding its ingress on the surface of the medium. After 5 days of incubation, the inoculated explants were washed three times with agrobacteria with sterile water and placed on a Hamburg B ₅ agar medium of the same composition, but with the addition of 500 mg / l cefotaxime and 50 mg / l kanamycin. Subculture on fresh medium was carried out every 3-4 weeks, while claforan was added to the medium, gradually reducing its concentration from 500 mg / l to 0 mg / l until the complete elimination of the agrobacterium. Kanamycin at a concentration of 50 mg / L was added throughout the entire process of transformation and maintaining the in vitro collection of transformed morphogenic cultures and shoots. DNA was isolated from young aseptic leaves of regenerated plants that formed roots in vitro in the presence of 50 mg / L kanamycin using the Edwards method with preliminary freezing at -20 ° C. PCR analysis was performed with primers manufactured by SYN-TOL.

Example 1

Genetic transformation of clover plants of the meadow varietal Early 2.

For genetic transformation, meadow clover genotypes with high PC were used, which showed high resistance to aluminum ions when evaluated in vitro, in growing and field experiments. 20 morphogenic cultures of each genotype with small shoots, cut into pieces of 3-5 mm in size, were placed with a slice up on Hamburg B ₅ medium with 2 mg / L BAP and were inoculated with an overnight culture of agrobacteria. In the experiments, we used the Agrobacterium tumefaciens LGV 3850 strain and A. rhizogenes A4 strain containing the vector plasmids pK22ac and pK22rs with the npt11 marker gene (as-ap and rs-ap genes are synthesis genes for amaranth and radish defensins that increase resistance to root rot). After 5 days, the inoculated morphogenic cultures were washed three times with sterile distilled water. The excess water was removed by blotting the morphogenic cultures between the layers of sterile filter paper, and then they were placed on a fresh agar medium of the same composition, but with the addition of 50 mg / L kanamycin (a selective factor for selecting cell cultures of meadow clover with the npt11 genes integrated) and 500 mg / l cefotaxime to inhibit the growth of agrobacteria. Kanamycin was added to the medium throughout the entire process of transformation and maintaining the in vitro collection of transformed morphogenic cultures of meadow clover. Subculturing on cefotaxime media was carried out every 3-4 weeks, gradually reducing its concentration to 0 mg / l until the complete elimination of the agrobacterium. Figure 1 presents a top view of green regenerant plants with and without roots, figure 2 is the same, the view from the back, in figure 3 chlorophyll-defective shoots.

Example 2

PCR - analysis of the presence of embedded genes.

Confirmation of the presence of the nptII (marker) and ac-ar (target) genes in the resulting plants was determined by PCR. Moreover, a very important point is that the plants were grown on Hamburg medium without the addition of claforan (pefotaxime), which excluded from the analysis non-transgenic plants that give a positive PCR result due to residual contamination with agrobacteria.

Due to the fact that the studied genes may contain their own peptide antibiotic genes with DNA sequences homologous to the introduced transgenes, we used primers to identify transgenic plants by PCR that are complementary to the promoter region of the 35S cauliflower mosaic virus and the terminal portion of the as-ar gene.

Plants were analyzed from the following experimental options.

No. 1 LGVac2 No. 2 LGVac15П No. 3 A4rsI No. 4 A4rs6 No. 6 A4acI200 No. 7 A4rs21 I No. 8 A4rs21 II No. 9 A4rs6 I No. 10 P8 - control, non-transformed plant.

Genomic DNA for analysis was isolated from the leaf tissue of aseptically grown plants by the Edwards method with preliminary freezing at -20 ° C. For PCR analysis, primers manufactured by SYNTOL were used.

In FIG. Figure 4 presents the results of a PCR analysis of meadow clover plants with npt1 / npt2 primers and plasmid pK22rs (annealing at 50 ° C) wells: 1 and 14 molecular weight marker; 2-water, 3-plasmid pK22rs, 4 - plasmid pK22rs + DNA of the original sample (control-P8), 5 - DNA of the original sample P8; 6-13 - DNA of transforming plants (No. 1, 2, 3, 4, 6, 7, 8, 9).

PCR analysis with the primer npt1 / npt2 at an annealing temperature of 50 ° C showed the amplification of all tested samples, except No. 7, with a single specific fragment at the level of luminescence of the plasmid, size 580-590 nucleotides. In the control sample of the source plant (lane No. 5 in FIG. 4), amplification was not observed.

Figure 5 presents the results of PCR analysis with primers 35S / ac meadow clover plants transformed with a strain containing a vector with the ac gene (annealing 52 ° C) wells: 1 - molecular weight marker, 2 - water, 3 - plasmid pK22ac, 4 - pK22ac + DNA of the initial sample (control - P8), 5 - DNA of the initial sample (P8), 6-8 DNA of transforming plants (Nos. 1, 2, 6).

By PCR analysis of the samples for the presence of the target amaranth defensin gene (ac), it was found that using a combination of 35S / ac primers at an annealing temperature of 52 ° C, amplification of samples transformed with the agrobacterium strain with the amaranth defensin gene was observed in the absence of amplification in the control non-transgenic plant ( 5 - lanes 6, 7, 8, samples LGVac2; LGVac15P; A4acl 200).

Thus, the presence of embedded genes in the genome of the studied meadow clover samples was shown.

Example 3

Acid resistance of morphogenic cultures of meadow clover on Hamburg's agarized nutrient medium B ₅ with 50 mg / l aluminum ions.

All transformed morphogenic cultures of meadow clover retained a sign of acid resistance on a Hamburg B ₅ agar nutrient medium after 50 mg / L of aluminum ions after long-term (for more than 56 passages) cultivation on a medium with selective factor kanamycin. The results are presented in the table.

Table Acid resistance of transgenic morphogenic cultures of meadow clover Genotype Indicators of resistance to 50 mg / l Al ³⁺ Average weight, mg% Average length, mm% shoots the roots shoots the roots source transgenic source transgenic source transgenic source transgenic A4rsK711-2

A4rs21 I

LGVac15P

LGVac2

The studied clones of 4 meadow clover genotypes retained the resistance to Al ³⁺ at the level of the initial forms, and the clones of 3 genotypes A4rs21 I, LGVac15P, LGVac2 even significantly exceeded them in terms of the mass of the formed roots 72.1; 54.6 and 125% respectively. Moreover, the root length was greater only in two genotypes (LGVac15P and LGVac2). A significant excess of the mass of morphogenic tissue was observed in clones A4rs21 I (93.4%). The average mass of shoots of transgenic clones exceeded that of the original samples in 3 genotypes A4rsK7 11-2, A4rs21 I and LGVac15P by 56.9; 38.5 and 32.2%, respectively.

Thus, the obtained data, including the ability to rhizogenesis of the studied clones on a selective medium, indicate the preservation of the sign of acid resistance by regenerant plants in the process of genetic transformation.

The developed method for the genetic transformation of plants of valuable breeding specimens of meadow clover allows one to obtain regenerant plants with built-in genes - marker (kanamycin resistance) and target (amaranth defensin synthesis gene that increases plant resistance to root rot).

In addition, the use of a direct plant regeneration method, which eliminates the occurrence of somaclonal variation, during genetic transformation allows 1) to maintain selection-valuable traits of the initial sample (acid resistance), 2) to maintain more than 50 passages in transgenic morphogenic cultures with high regenerative ability in vitro culture, 3 ) receive an unlimited number of transgenic regenerated plants.

The application of the inoculum only on the cut surface facilitates subsequent procedures for the elimination of agrobacterial infection of morphogenic tissue.

Using for PCR analysis of DNA isolated from the leaf tissue of aseptic regenerated plants that formed roots on a medium with 50 mg / l kanamycin, it is possible to detect transgenic plants at earlier stages of cultivation before planting in the soil.

Information sources

1. Solodkaya L.A., Agafodorova M.N., Kurenina L.V., Lapotyshkina L.V. Method for the regeneration of meadow clover plants during genetic transformation. / Patent for invention RU No. 2305931, 2007

2. Michael L. Sullivan, Kenneth H. Quesenberry Red Clover (Trifolium pratense) // Methods in Molecular Biology, vol. 343: Agrobacterium Protocols, 2 / e, volume 1. Edited by: Kan wang, Humana Press Inc., Totowa , N1, p. 369-383.

Claims (1)

A method for the genetic transformation of plants of breeding valuable samples of meadow clover, including inoculating the surface of explants placed on Hamburg B ₅ medium without antibiotics, agrobacterial culture, cultivation for 48 h, further cultivation of explants washed from agrobacteria on Hamburg B ₅ medium with 50 mg / l kanamycin in the presence of an antibiotic eliminating an agrobacterium, plant regeneration and PCR analysis of the presence of integrated genes, characterized in that the inoculation is carried out by applying an agrobacter with a spatula on the cut surface of parts 3-5 mm in size of morphogenic tissue with shoots placed on Hamburg B ₅ medium with 2 mg / l of 6-benzylaminopurine, followed by in vitro regeneration of plants by direct regeneration, without the formation of callus tissue, and PCR analysis DNA isolated from young aseptic leaflets of in vitro regenerated plants, which formed roots in the presence of a selective factor of kanamycin at a concentration of 50 mg / l, and control the conservation of selection-valuable (acid resistance) traits of the initial sample.

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