RU2619175C1 - Method for genetically modified trees production - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method includes: reception of plant explants of Agrobacterium tumefaciens straib and their preparation for inoculation; reception of plant explants and their preparation for inoculation; explant inoculation; explant co-cultivation; explants washing from agrobacteria; explant placing on a medium containing selective agents and bacteriostatic antibiotics; selection of the obtained regenerants on the selective media. The said method is characterized by plant explants preparation for inoculation, explants inoculation with agrobacteria and explants washing from agrobacteria are performed in a culture medium containing all components of the medium for co-cultivation, selection and regeneration, with the exception of gelling agents, plant growth regulators, bacteriostatic antibiotics or selective agents.

EFFECT: application of this method prevents osmotic stress in transformed plant cells and increases the frequency of genetic transformation of plants.

2 cl, 3 dwg, 1 tbl, 6 ex

Description

The invention relates to the field of plant biotechnology using genetic engineering methods, and can be used to obtain genetically modified woody plants expressing heterologous genes.

Woody plants related to both fruit crops and forest species are of great economic importance. Fruit harvests in Russia are estimated in millions of tons, and harvesting volumes by tens of millions of cubic meters. The appearance of genetically modified trees with new traits that cannot be given by traditional breeding methods can significantly increase the efficiency of both agriculture and forestry. However, due to the biological characteristics of woody plants, the efficiency of their genetic transformation is significantly lower than that of herbaceous plants.

The first transgenic forest species, a poplar-aspen hybrid, was obtained in 1987 (Fillatti JJ, Sellmer J., McCown B., Haissig B., Comai L. Agrobacterium mediated transformation and regeneration of Populus. Mol. Gen. Genet. 1987. 206: 192-199), and the first transgenic fruit crop, the apple tree, in 1989 (James DJ, Passey AJ, Barbara DJ Genetic transformation of apple (Malus pumila Mill.) Using a disarmed Ti-binary vector. Plant Cell Rep. 1989. 7: 658-661), that is, just a few years after receiving the first transgenic plants in 1983. However, at present, woody transgenic plants are disproportionately less distributed than grassy ones. In 2014, transgenic plants occupied more than 181 million ha in the world (James C. 2014. Global Status of Commercialized Biotech / GM Crops: 2014. IS AAA Brief No. 49. ISAAA: Ithaca, NY), and of which the share of transgenic trees , poplars and papaya, accounted for, at best, only tens of thousands of hectares. Last but not least, this state of affairs is associated with the low efficiency of transformation of woody plants.

The efficiency (frequency) of transformation is calculated as the ratio of the number of primary transgenic plants (for which the presence of an insert in the genome is confirmed) to the number of explants used for transformation, and is presented as a percentage. The transformation of woody plants in most cases (with the exception of conifers) is carried out in an agrobacterial way and a large number of factors related to both the plant and its cultivation, as well as agrobacteria can influence its effectiveness. These factors include the genotype of the plant, the type of explant, its physiological state, the composition of the nutrient medium, the bacterial strain and / or vector, and several others.

The standard process of genetic transformation of plant explants using agrobacteria includes several stages: preparation of explants, inoculation of explants in agrobacterial suspension, cocultivation of explants with agrobacteria, washing of explants from excess agrobacteria, and regeneration of plants from explants on selective medium. The stages of co-cultivation and regeneration take place on solid nutrient media, which, as a rule, contain the same set of mineral salts, carbohydrates, plant growth regulators and differ only in the content of bacteriostatic antibiotics and selective agents (they are absent in the media for co-cultivation). All other manipulations with explants (preparation, inoculation, washing) are carried out in a liquid, which is water or a liquid nutrient medium. It is known that the mineral salts and carbohydrates that make up the nutrient medium influence the growth of plant cells, not only because they are a source of nutrition for them, but also due to their osmotic properties. Thus, the periodic placement of plant explants in the process of genetic transformation under conditions with different osmotic pressure can cause osmotic stress in plant cells, thereby reducing their viability and, consequently, the ability to genetic transformation and subsequent regeneration into whole plants. This is especially important for explants of woody plants, which are characterized by a fairly low frequency of transformation. To prevent the negative impact of osmotic stress, plant explants should be kept during the entire process of genetic transformation under similar osmotic conditions.

Known methods of genetic transformation of woody plants in which water is used to prepare an agrobacterial suspension (Matsunaga E., Sugita K., Ebinuma N. An asexual production of selectable marker-free transgenic woody plants, vegetatively propagated species. Mol Breed. 2002. 10: 10: 95-106) or washing off excess agrobacteria (US Patent 5,922,928. Chiang et al. An Agrobacterium-mediated transformation and regeneration method for plants including a transformation method to produce transgenic plants with an altered lignin composition. July 13, 1999). The disadvantage of these methods is that the chemically pure (distilled) water used in them has the highest water potential, which is very different from the water potentials of the used culture media for cocultivation / regeneration - MS (Murashige T., Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 1962.15: 473-497) or WPM (Lloyd G., McCown BH Commercially feasible micropropagation of mountain laurel, (Kalmia latifolia) by use of shoot tip culture. Int. Plant Prop Soc., Comb. Proc. 1980. 30: 421-427), respectively.

K., Pappinen A., von Weissenberg K. Comparisons of the efficiency of some promoters in silver birch (Betula pendula). Plant Cell Rep. 1998. 17: 356-361) применяли среду MS. Недостатком этих способов является то, что минеральный состав питательных сред для инокуляции и отмывки отличался от состава сред для кокультивации/регенерации. Кроме того, среды для инокуляции и отмывки по сравнению со средами для кокультивации/регенерации или не содержали углеводов вообще (Aldwinckle et al., 2000;

et al., 1998), или содержали их в 3 с лишним раза более высокой концентрации (Matsuda et al., 2005).Genetic transformation methods are also known in which various liquid nutrient media are used to prepare an agrobacterial suspension or to wash off excess agrobacteria. For example, for the preparation of an agrobacterial suspension during transformation of pome seeds (US Patent 6,100,453. Aldwinckle et al. Transgenic pomaceous fruit with fire blight resistance. August 8, 2000), 1/2 MS medium was used, and for pear transformation (Matsuda N., Gao M. , Isuzugawa K., Takashina T., Nishimura K. Development of an Agrobacterium-mediated transformation method for pear (Pyrus communis L.) with leaf-section and axillary shoot-meristem explants. Plant Cell Rep. 2005. 24: 45-51 ) - 1/10 NN medium (Nitsch JP, Nitsch C. Haploid plants from pollen grains. Science. 1969. 163: 85-87). To wash explants from excess agrobacteria during birch transformation (

K., Pappinen A., von Weissenberg K. Comparisons of the efficiency of some promoters in silver birch (Betula pendula). Plant Cell Rep. 1998.17: 356-361) MS medium was used. The disadvantage of these methods is that the mineral composition of the culture media for inoculation and washing was different from the composition of the media for cocultivation / regeneration. In addition, media for inoculation and washing compared to media for cocultivation / regeneration or did not contain carbohydrates at all (Aldwinckle et al., 2000;

et al., 1998), or contained them more than 3 times higher concentration (Matsuda et al., 2005).

J., Jacobsen H.J., Kiesecker H. Transformation of apple (Malus domestica Borkh.) with the stilbene synthase gene from grapevine (Vitis vinifera L.) and a PGIP gene from kiwi (Actinidia deliciosa). Plant Cell Rep. 2003. 22: 141-149), в котором для инокуляции, отмывки, кокультивации/регенерации использовалась одна и та же среда - MS. Недостатком ближайшего аналога является то, что среды для инокуляции и отмывки не содержали углеводов, тогда как среда для кокультивации/регенерации содержала 3% сорбитола. Кроме того, перед однократной отмывкой эксплантов от избытка агробактерий средой MS проводилась их двукратная отмывка водой, что подвергало растительные клетки воздействию осмотического стресса.Closest to the claimed invention is a method for the genetic transformation of an apple tree (Szankowski I., Briviba K., Fleschhut J.,

J., Jacobsen HJ, Kiesecker H. Transformation of apple (Malus domestica Borkh.) With the stilbene synthase gene from grapevine (Vitis vinifera L.) and a PGIP gene from kiwi (Actinidia deliciosa). Plant Cell Rep. 2003. 22: 141-149), in which the same medium was used for inoculation, washing, cocultivation / regeneration - MS. A disadvantage of the closest analogue is that the media for inoculation and washing did not contain carbohydrates, while the media for cocultivation / regeneration contained 3% sorbitol. In addition, before washing the explants once from excess agrobacteria with MS medium, they were twice washed with water, which exposed the plant cells to osmotic stress.

The objective of the invention is to increase the frequency of genetic transformation by maintaining plant explants during the whole process in conditions with similar osmotic properties.

The problem is solved due to the fact that in the method of genetic transformation, including: a) obtaining and preparing for inoculation of plant explants of the Agrobacterium tumefaciens strain containing selective, marker and / or useful genes intended for introduction into the plant; b) obtaining and preparing for inoculation with strain A. tumefaciens of plant explants; c) inoculation of explants with A. tumefaciens strain in a liquid medium; d) cocultivation of explants with agrobacteria on a medium without selective agents and bacteriostatic antibiotics; e) washing the explants from agrobacteria; f) placing the explants on a medium containing selective agents and bacteriostatic antibiotics, in order to obtain regenerants directly from the tissues of the explants or through the callus stage; g) selection of regenerants obtained on a selective medium by testing them for the presence of a full-sized copy of the target gene, the following difference is provided - preparation of plant explants for inoculation in a culture medium containing all components of the medium for cocultivation, selection and regeneration, with the exception of gelling agents, growth regulators plants, bacteriostatic antibiotics and selective agents, as well as additionally, but not necessarily, substances that serve as attractants for agrobacte s, e.g. Acetosyringone. In addition, the proposed method is characterized in that: a) the inoculation of explants with agrobacteria is carried out in a culture medium containing all components of the medium for cocultivation, selection and regeneration, with the exception of gelling agents, plant growth regulators, bacteriostatic antibiotics and selective agents; b) washing of explants from agrobacteria is carried out in a culture medium containing all components of the medium for cocultivation, selection and regeneration, with the exception of gelling agents, plant growth regulators and selective agents; c) woody plants are selected from the group including apricot, quince, cherry plum, cherry, pear, peach, plum, cherry, apple, birch, beech, elm, willow, chestnut, maple, linden, aspen, poplar, ash.

The implementation of the invention

Examples in the detailed description are given for the stock of pear GP 217, the birch bp3f1 genotype and the aspen Pt genotype.

However, the list of plants for use in this invention is not limited to these plants.

In particular, in vitro material is used as explants for transformation: pear and birch leaves, aspen internodes.

Examples in the detailed description are provided for the gus marker gene.

The possibility of implementing the proposed method is confirmed by the presented examples, but is not limited to them.

Example 1. Preparation of bacterial strains of A. tumefaciens CBE21 (pCBE21, pBI121) for plant transformation

For the transformation of plants using a night culture of bacteria A. tumefaciens. For this, 100 μl of A. tumefaciens CBE21 bacterial cell suspension (pCBE21, pBI121) is added to 50 ml of LB liquid medium containing 50 mg / l kanamycin and incubated overnight on a thermostatic orbital shaker at 28 ° C and 120-150 rpm min, after which the resulting suspension is centrifuged for 5 minutes at 4000 rpm, the precipitate is washed with liquid MS and centrifugation and washing are repeated. After cell precipitation, the supernatant is drained and 50 ml of liquid MS without carbohydrates (standard) or liquid NN, MS, MSm (modified MS medium containing 10 mM NH ₄ NO ₃ and 30 mM KNO ₃ ) containing 30 g / l sucrose (options for the transformation of pears, birch, aspen, respectively), after which they are resuspended.

Example 2. Transformation of pear plants by bacterial cells of A. tumefaciens CBE21 (pCBE21, pBI121)

A) Preparation of plant material

To transform pear plants using leaf explants from rooted plants in vitro. Propagation of the pear culture is carried out on a nutrient medium containing mineral salts QL (Quoirin M, Lepoivre P. Improved medium for in vitro culture of Prunus sp. Acta Hort. 1977. 78: 437-442), vitamins JS (Jacobini A., Standardi A La moltiplicazione in vitro del melo cv. Wellspur. Rivisia della Ortoflorofrutticoltura Italiana. 1982. 66: 217-229), 1.5 mg / L BAP, 0.2 mg / L IMA, 0.3 mg / L HA, 100 mg / l of mesoinositis, 20 g / l of sucrose and 7 g / l of agar. Rooting of pear shoots is carried out on a nutrient medium containing mineral salts QL with a halved content of NH ₄ NO ₃ , vitamins MS, 0.5 mg / L IMC, 10 g / L sucrose and 7 g / L agar. Plants are grown at a photoperiod of 16/8 hours, a temperature of 22-24 ° C and illumination of 3000-3500 lux.

B) Genetic transformation of explants of pear plants

For transformation, leaves from rooted plants that were on the rooting medium for 1.5-2 months are used. The leaves are cut and placed in Petri dishes containing either water (standard version) or liquid NN medium with 30 g / l sucrose. Petioles and apices are removed from the leaves (large leaves also have lateral sides) and several incisions are made perpendicular to the central vein, without bringing them to the edges of the sheet. The explants prepared in this way are placed for 40-50 minutes in a suspension of agrobacteria, then they are dried with sterile paper filters and placed on paper filters located in Petri dishes on the surface of the cultivation medium containing mineral salts NN, vitamins JS, 3 mg / L TDZ, 0.5 mg / L NAA, 0.1 mg / L HA, 100 mg / L Mesoinositol, 100 mg / L Casein Hydrolyzate, 30 g / L Sucrose and 7 g / L Agar. 10-15 explants are placed in each cup. Cocultivation of explants with agrobacteria is carried out in the dark at a temperature of 23 ° C for three days. After cocultivation, the explants are washed from excess agrobacteria either in water containing 500 mg / l cefotaxime (standard option) or in liquid NN medium containing 30 g / l sucrose and 500 mg / l cefotaxime. Washed explants are dried on paper filters and transferred to medium for regeneration and selection of transformants of the same composition as the medium for cocultivation, containing an additional 25 mg / l kanamycin and 500 mg / l cefotaxime. On this medium, the explants are maintained for 4-6 subcultures of four weeks in the dark at a temperature of 23 ° C. The regenerated shoots are transplanted onto a propagation medium containing 25 mg / L kanamycin and 250 mg / L cefotaxime.

Example 3. Transformation of birch plants by bacterial cells of A. tumefaciens CBE21 (pCBE21, pBI121)

A) Preparation of plant material

To transform birch plants, leaf explants from plants in vitro are used. Propagation of the birch culture is carried out on a WPM nutrient medium containing 0.6 mg / L BAP, 0.1 mg / L IMC, 20 g / L sucrose and 7 g / L agar. Plants are grown at a photoperiod of 16/8 hours, a temperature of 22-24 ° C and illumination of 3000-3500 lux.

B) Genetic transformation of explants of birch plants

For transformation use leaves from plants in vitro, one month old. The leaves are cut and placed in Petri dishes containing either water (standard version) or liquid MS medium with 30 g / l sucrose. Petioles and apices are removed from the leaves (large leaves also have lateral sides) and several incisions are made perpendicular to the central vein, without bringing them to the edges of the sheet. Thus prepared explants are placed for 40-50 minutes in a suspension of agrobacteria, then they are dried with sterile filters and placed on filters located in Petri dishes on the surface of the cultivation medium containing MS mineral salts, 5 mg / L zeatin, 5 mg / L BAP , 0.2 mg / l IMA, 30 g / l sucrose and 7 g / l agar. 10-15 explants are placed in each cup. Cocultivation of explants with agrobacteria is carried out in the dark at a temperature of 23 ° C for three days. After cocultivation, the explants are washed from excess agrobacteria either in water containing 500 mg / l cefotaxime (standard option) or in liquid MS medium containing 30 g / l sucrose and 500 mg / l cefotaxime. The washed explants are dried on paper filters and transferred to a medium for regeneration and selection of transformants of the same composition as the cocultivation medium containing an additional 50 mg / l kanamycin and 500 mg / l cefotaxime. On this medium, the explants are kept under 16-hour daylight hours at 22-23 ° C with a transplant every 4 weeks. The regenerated shoots are transplanted onto a propagation medium containing 50 mg / l kanamycin and 250 mg / l cefotaxime.

Example 4. Transformation of aspen plants by bacterial cells of A. tumefaciens CBE21 (pCBE21, pBI121)

A) Preparation of plant material

In vitro plant internodes are used to transform aspen plants. Propagation of the aspen culture is carried out on a hormone-free nutrient medium WPM containing 1 mg / l of MS vitamins, 30 g / l of sucrose and 7 g / l of agar. Plants are grown at a photoperiod of 16/8 hours, a temperature of 22-24 ° C and illumination of 3000-3500 lux.

B) Genetic transformation of explants of aspen plants

For transformation, internodes of plants 3-4 weeks old are used, cut into segments of 5-10 mm. The explants prepared in this way are placed in a suspension of agrobacteria for 30 minutes, after which they are dried with sterile filters and placed on filters located in Petri dishes on the surface of the cultivation medium containing MSm mineral salts, MS vitamins, 0.5 mg / L, 2.4- D, 30 g / l sucrose and 7 g / l agar. 20 explants are placed in each dish. Cocultivation of explants with agrobacteria is carried out in the dark at a temperature of 23 ° C for three days. After cocultivation, the explants are washed from excess agrobacteria either in water containing 500 mg / l cefotaxime (standard option) or in liquid MSm containing 30 g / l sucrose and 500 mg / l cefotaxime. Washed explants are dried on paper filters and transferred to a medium for regeneration and selection of transformants of the same composition containing instead of 0.5 mg / L 2,4-D 0.5 mg / L Zeatin and an additional 30 mg / L Kanamycin and 500 mg / l cefotaxime. On this medium, the explants are kept under 16-hour daylight hours at 22-23 ° C with a transplant every 4 weeks. The regenerated shoots are transplanted onto a propagation medium containing 30 mg / L kanamycin and 250 mg / L cefotaxime.

Example 5. Polymerase chain reaction (PCR) to the presence of a fragment of the sequence of the gus gene

The presence of the gus gene in transgenic pear, birch, and aspen plants is confirmed by PCR with 35S primers (SEQ ID NO: 1) and uidA-low (SEQ ID NO: 2) specific for the CaMV 35S promoter sequence and the interior of the marker gene.

Genomic DNA from pear, birch and aspen plants was isolated by the method of Rogers and Bendich (Rogers SO, Bendich AJ Extraction of total cellular DNA from plants, algae and fungi. In: Plant Molecular Biology Manual. Gelvin SB, Schilperoort RA (Eds.). Kluwer Academic Publisher, 1994. D1: 1-8). For isolation, plant leaves are used in vitro (about 100 mg). The resulting plant DNA is used as a template in PCR assays. The reaction mixture contains 16 mM (NH ₄ ) ₂ SO ₄ , 0.01% BSA, 200 μM of each dNTP, 0.4 μM of each oligonucleotide, 0.05 units / μl of Taq polymerase, 1-5 ng / μl of genomic DNA. The reaction is carried out in a volume of 50 μl under the following conditions: 96 ° C for 3 minutes; 30 cycles: 94 ° C - 1 min, 60 ° C - 1 min, 72 ° C - 1 min, then 72 ° C - 5 min.

PCR products are analyzed on a 1.8% agarose gel with the addition of ethidium bromide. The gel is photographed in ultraviolet at a wavelength of 260-280 nm. The appearance of the PCR product (DNA with a size of 1149 bp) when using these primers, as well as in the absence of it in the reactions put on control DNA, indicates the presence of the desired gene in the DNA of the studied plants (Fig. 1, Fig. 2 and . 3).

Example 6. Comparison of various methods of genetic transformation

The genetic transformation of pear, birch and aspen with the gus gene is carried out either in the standard way, when the explants are prepared and washed from agrobacteria in water, and inoculation in the MS liquid medium, or in a modified way, when the preparations, inoculation, and washed pear explants are in NN liquid containing 30 g / l sucrose, explants of birch in MS liquid medium containing 30 g / l sucrose, and aspen explants in MSm liquid medium containing 30 g / l sucrose. The regenerates obtained from explants of the corresponding variants are checked for the presence of gus gene sequences, as described in Example 5. The results of the transformation in various ways are presented in Table 1.

From the presented data, it can be concluded that there is a causal relationship between the maintenance of explants of woody plant species during genetic transformation under conditions with similar osmotic properties, which prevents osmotic stress, and the effectiveness of genetic transformation. An increase in the frequency of transformation under the above conditions was observed for various species of woody plants, which indicates the universality of the proposed method.

Thus, the invention allows to increase the efficiency of genetic transformation of woody plants by preventing osmotic stress in explant cells due to their presence during the entire process of genetic transformation in conditions with similar osmotic properties.

Claims (2)

1. A method of producing genetically modified woody plants, including: a) obtaining and preparing for inoculation of plant explants of the Agrobacterium tumefaciens strain containing selective, marker and / or useful genes intended for introduction into the plant; b) obtaining and preparing for inoculation with strain A. tumefaciens of plant explants; c) inoculation of explants with A. tumefaciens strain in a liquid medium; d) cocultivation of explants with agrobacteria on a medium without selective agents and bacteriostatic antibiotics; e) washing the explants from agrobacteria; f) placing the explants on a medium containing selective agents and bacteriostatic antibiotics, in order to obtain regenerants directly from the tissues of the explants or through the callus stage; g) selection of regenerants obtained on a selective medium by testing them for the presence of a full-sized copy of the target gene, characterized in that the preparation of plant explants for inoculation, inoculation of explants with agrobacteria and washing of explants from agrobacteria are carried out in a culture medium containing all components of the medium for cocultivation, selection and regeneration, with the exception of gelling agents, plant growth regulators, bacteriostatic antibiotics and selective agents, as well as additionally containing, but not necessarily substances that serve as attractants for agrobacteria, such as acetosyringone. 2. The method according to p. 1, characterized in that the woody plants are selected from the group comprising apricot, quince, cherry plum, cherry, pear, peach, plum, cherry, apple, birch, beech, elm, willow, chestnut, maple, linden , aspen, poplar, ash.

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