KR20180085153A - Method of enhancing resistance against high salt stress of plant - Google Patents

Abstract

The present invention provides a method for enhancing salt resistance of rice, which comprises the following steps: a neglect step for disinfecting and washing full mature seeds of the rice and neglecting the full mature seeds of the rice in a medium at the temperature of 24 to 27°C and under a light-free condition for 5 to 6 weeks; and a gradual culture step for culturing a callus selected in the neglect step at the temperature of 24 to 27°C and under the light-free condition while gradually increasing the salinity of salts. According to one embodiment of the present invention, it is possible to cultivate a plant under a high salt stress condition to realize effective salt-resistance increasing effect resistant to salt stress.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for increasing the salt tolerance of a plant,

The present invention relates to a method for increasing the salt tolerance of a plant, and more particularly, to a method for increasing the salt tolerance by culturing a plant under high salt stress conditions.

Plants show different responses to different stresses on different species. Due to the difference in these responses, various plant species are classified as endogenous and susceptible species for various stresses. In food production, drought and salt are major abiotic stresses causing significant reductions in crop quantity and quality each year. Drought is usually accompanied by salt, and salinization of the soil is a common soil deterioration phenomenon in drought areas. to be. It is important to clarify the effects of salinity stress on the plants because salinity stress can be caused not only by the problems in the reclaimed soil but also by the salt accumulation in the soil by a pair of soils. Salinity accumulation in the soil is problematic not only in the existing vegetation-free soils such as reclaimed land, but also in the facilities. Salinity stress is mainly caused by the accumulation of sodium chloride in the soil. The response of plants to these stresses is attempting to investigate the mechanisms of salinity stress and tolerance mechanisms based on the difference in response between the saliva plants and the saline plants. It is necessary to develop plants with tolerance to salt tolerance even in a high salt stress environment. In this regard, Korean Patent Registration No. 0943414 discloses a method for increasing drought and salt resistance of a plant and a plant therefrom.

However, in the case of the prior art, 2R, 3R-butanediol, which is not a salt to a plant, is treated to a plant, which has a problem of low salting efficiency.

The present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide a method for culturing a plant under high salt stress conditions to increase salt resistance more efficiently than the conventional method. However, these problems are exemplary and do not limit the scope of the present invention.

 According to one aspect of the present invention, an organic step of disinfecting and washing the rice seeds, followed by allowing the seeds to stand in a medium at a temperature of 24-27 캜 and under a light-free condition for 5 to 6 weeks; And a step-culturing step of culturing the callus selected in the organic step at a temperature of 24-27 DEG C and in the absence of light in a stepwise increasing concentration of the salt. A method for increasing the salt tolerance of rice is provided.

According to one embodiment of the present invention as described above, an effective salt tolerance increase effect that is resistant to salt stress can be realized by culturing a plant under a high salt stress condition. Of course, the scope of the present invention is not limited by these effects.

FIG. 1 is a photograph showing a process of inducing a salt adaptive plant according to an embodiment of the present invention.
FIG. 2 is a graph showing a salt adaptation process of callus according to an embodiment of the present invention. FIG.
FIG. 3 is a graph showing the germination percentage (A) and the length of the top part (B) of a general rice cultivar in MS (Murashige & Skoog) medium containing salt according to an embodiment of the present invention.
FIG. 4 is a photograph showing germinated seeds (R1 seed) harvested from regenerated plants according to an embodiment of the present invention after culturing in an MS medium.
FIG. 5 is a graph showing the germination percentage after culturing seeds (R1 seed) harvested from regenerated plants in an MS medium according to an embodiment of the present invention.
FIG. 6 is a photograph showing the salt stress tolerance of callus after cell culture in a salt-adapted seed (R1 seed) according to an embodiment of the present invention.
FIG. 7 is a graph showing fresh weight of callus after cell culture in a salt-adapted seed (R1 seed) according to an embodiment of the present invention.
FIG. 8 is a graph showing chlorophyll contents of the R1 plant germinated from a salt-adapted seed (R1 seed) according to an embodiment of the present invention.
FIG. 9 is a graph showing an analysis of potassium ion content (Ion contents) of a seedling (R1 plant) germinated from a salt-adapted seed (R1 seed) according to an embodiment of the present invention.
10 is a graph showing sodium ion contents (Ion contents) of seedlings (R1 plant) germinated from a salt-adapted seed (R1 seed) according to an embodiment of the present invention.
11 is a graph showing an analysis of calcium ion contents (Ion contents) of seedlings (R1 plant) germinated from a salt-adapted seed (R1 seed) according to an embodiment of the present invention.
FIG. 12 is a graph illustrating the K ± / Na ± ratio calculated from the ion content measurement result according to an embodiment of the present invention.
FIG. 13 is a photograph showing the comparison of the growth state of rice plants germinated from R1 seeds after salt treatment in a greenhouse according to an embodiment of the present invention.
FIG. 14 is a photograph showing a salt tolerance enhancement in an R2 plant germinated from an R2 seed harvested from an R1 plant according to an embodiment of the present invention. FIG. 14 is a photograph (A) It is a graph (B).

Definition of Terms:

As used herein, the term " salt tolerance " refers to the resistance of a plant to high salt concentrations in the soil. Salinity tolerance occurs in seawater intrusion zones, high temperature dry sites, and plantations with high salt concentrations. At high salt concentrations, plants are normally inhibited by water absorption inhibition by increased osmotic pressure and increased salinity in the body. However, plants that have an ability to increase the osmotic pressure corresponding to the change in osmotic pressure of the medium or accumulate excessive salts in the vacuole exhibit salt resistance, and the degree of this salt tolerance is somewhat different among plant species.

As used herein, the term "callus" is a tissue that occurs when a plant is injured. It may be cultured in a medium containing auxin by cutting off the plant tissue with oil phase, A wound or a wound) is treated or treated with auxin.

DETAILED DESCRIPTION OF THE INVENTION [

  According to one aspect of the present invention, an organic step of disinfecting and washing the rice seeds, followed by allowing the seeds to stand in a medium at a temperature of 24-27 캜 and under a light-free condition for 5 to 6 weeks; And a step-culturing step of culturing the callus selected in the organic step at a temperature of 24-27 DEG C and in the absence of light in a stepwise increasing concentration of the salt. A method for increasing the salt tolerance of rice is provided.

In the method for increasing the salt tolerance of the rice, the seed of the ripened seed may be the seed coat removed, and the salt may be NaCl.

In the method for increasing the salt tolerance of the rice, the concentration of the salt in the culturing step may be increased stepwise from 110 to 130 mM at 10 to 30 mM, and the salt concentration may be increased by 10 to 30 mM at each step, The steps may be performed for 3 to 5 weeks for each step.

Hereinafter, the present invention will be described in more detail by way of examples. It should be understood, however, that the invention is not limited to the disclosed embodiments, but may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, Is provided to fully inform the user.

Example 1: Cell culture and salt adaptation

According to one embodiment of the present invention, an embryogenic callus was obtained from the seed of the seedling of Oryza sativa (Milyang 122), which is a general cultivated rice, in CI (callus induction) medium (2N6-1, see Table 1) Organic calli were induced in the CI medium containing 20 mM NaCl to induce salt adaptation.

Specifically, the seed husks removed from the seeds were used for organizing the calli, and the rice huller used for hand-rubbing was used to remove the seeds. Then, the seeds from which the seed coat had been removed were sterilized once with 70% ethanol for 10 minutes, and then sterilized twice for 20 minutes by addition of Triton x-100 to 1.2% sodium hypochlorite solution and sterilized And washed more than 5 times with water. Seven disinfected seeds were then placed in a petridish of 9-10 cm in diameter, and then cultured at 25-26 ° C for 5-6 weeks in the absence of light. Of the calli cultured in the CI medium, pale yellow calli of 2-3 mm in diameter were selected except for friable mucinous calli, and some were cultured in the same medium supplemented with cell culture or 20 mM NaCl The cells were allowed to stand in a CI medium at 25-26 DEG C for 4 weeks without light. Then, the cells were cultured in a medium containing 20 mM NaCl to prepare a callus of embryogenic callus (2-3 mm in diameter) of soft yellow color with a smooth cell proliferation and no browning. ) Were selected and allowed to stand under the same conditions for 4 weeks for CI medium supplemented with 40 mM NaCl. In the same manner, the callus cultured in the medium supplemented with 40 mM NaCl was allowed to stand for 4 weeks in the CI medium supplemented with 60 mM NaCl. Some of the calli cultured in the medium supplemented with 60 mM NaCl were partly grown in the same medium CI medium supplemented with 80 mM NaCl was left for 4 weeks under the same conditions. Likewise, some of the calli cultured in the medium supplemented with 80 mM NaCl were allowed to stand in the same medium and the rest in the CI medium supplemented with 100 mM NaCl for 6-7 weeks. Then, 100 mM NaCl was added to the culture medium Some of the calli were incubated in CI medium supplemented with 120 mM NaCl for 6-7 weeks.

A0 (60 mM), A80 (80 mM), A100 (100 mM), and A120 (100 mM) were determined according to the salt concentrations adjusted for each salt concentration. 120 mM adaptation) (Fig. 1). The composition of the medium used for inducing the CI medium and the callus regenerating individuals described below is shown in Table 1 below.

Cl
(Callus induction & adaptation)
2N6-1 medium PRN
(Pre-reproduction-I)
N6-7-CH medium RN-I
(Playback-I)
N6S3-CH-I medium RN-II
(Regeneration-II)
N6S3-CH-II medium RI
(Root induction)
MS badge N6 salt 1X N6 salt 1X N6 major salt 1X N6 Major salt 0.5X MS salt 1X B5 Minor salt 1X B5 Minor salt 1X MS Iron 1X MS Iron 1X N6 vitamin 1X N6 vitamin 1X B5 Vitamin 1X B5 Vitamin 1X MS Vitamin 1X Casamino acid 0.3 g / L Casamino acid 2g / L Casamino acid 2g / L Casamino acid 2g / L Sorbitol 30 g / L Sorbitol 30 g / L Proline 0.5 g / L NAA 1 mg / L NAA 1X Glutamine 0.5 g / L 6-benzyladenine 0.5 mg / L AA amino acid 1X AA amino acid 1X 2-4-D 2 mg / L 2-4-D 1 mg / L Kinetin 5mg / L Kinetin 2 mg / L Sucrose 3% Sucrose 2% Sucrose 2% Sucrose 3% Sucrose 3% Phytase 0.25% Phytase 0.25% Phytase 0.5% Phytase 0.25% Pita Gel 0.35% pH 5.7 pH 5.7 pH 5.7 pH 5.7 pH 5.7

Example 2 Induction of Seed Regeneration of Salt Adaptive Callus

In accordance with one embodiment of the present invention, induction of regeneration of salt-adaptive calli was allowed to proceed for 10 days under light-free conditions at 25-26 ° C for PRN (pre-regeneration) medium (N6-7-CH) ) Was induced to form green spots in the RN-I (regeneration-I) medium (N6S3-CH-I) at 25-26 캜 for 3 weeks under the long day condition (LD 16: 8h) . The callus in which the green body was formed was subjected to indirect shoot formation and shoot elongation at 25-26 ° C under long day conditions (LD 16: 8h) against RN-II medium (N6S3-CH-II) (Fig. 2C). Subsequently, the developed individuals were transferred to root induction medium (MS) for induction of root development at 25-26 ℃ under long-day conditions (LD 16: 8h) for 2-3 weeks, (Fig. 2D). All of the mediums (PRN, RN-I, RN-II, and MS) used until the regeneration was obtained were supplemented with NaCl at the same concentration as the salt concentration adapted for each cell line.

As a result, a lineage of the rice plant (Regeneration 0 generation) regenerated from the salt adaptive callus was secured and it is shown in Table 2 below.

Salt-adapted rice RO cell line R0A0 1-27 (27 plants) R0A60 1-38 (38 plants) R0A80 1-18 (18 plants) R0A100 1-9 (9 plants) R0A120 1-14 (14 plants)

Example 3: Investigation of R0 rice plant length and yield component

According to one embodiment of the present invention, the regrowth rice (R0) plant shown in Table 2 was purified for 3 weeks in a greenhouse and then packed into pavement, and the plant height was measured at the heading stage (about 80 days after growth) The number of seeds per panicle per m ² , spikelet per panicle, ripened grain, and 1000 (1000) grain counts, which determine the growth rate of rice, -grain weight and yield were calculated (statistical analysis was performed by SAS system at Duncan's multiple range test method at 5% significance level).

As a result, as shown in Table 3, the plant length of the regenerated rice seedlings was shorter than that of the control group, and the number of tillers was larger than that of the control group, Despite the fact that the seeds were not grown completely, the number of spikelets per unit area was higher than that of the seeds. The seeds of R0 generation (R1 seed) were obtained after harvesting, and the lines of regenerated rice obtained seeds are shown in Table 4 below.

lines Length (cm) Number of teeth
(per m ² ) Ripple per ear
(per panicle) Maturity rate
(%) Ginseng (g) yield
(Brown rice, kg / 10a) R0A0 64.0 + - 6.81b 206 ± 91.6 bc 51 ± 9.0b 70.8 ± 11.86b 23.9 ± 0.91abc 179.6 ± 88.46b R0A60 65.1 + - 10.04 b 206 ± 103.3 bc 55 ± 11.7b 65.2 ± 18.46b 25.4 + - 0.64a 196.4 + 134.87b R0A80 56.8 ± 8.12 cd 236 ± 123.0 bc 38 ± 9.9c 36.0 ± 22.52 c 22.9 ± 2.52 bcd 70.0 ± 53.30 cd R0A100 53.3 ± 3.09 d 475 ± 277.0a 34 ± 4.9 c 8.4 ± 4.42d 20.5 ± 6.90e 25.7 ± 14.77 d R0A120 61.3 ± 5.58 bc 298 ± 189.1b 54 ± 6.7b 42.6 ± 16.97 c 22.5 ± 1.05 cd 137.6 ± 83.05bc WT 75.2 ± 1.59a 176 ± 25.0 c 131 ± 9.9a 89.5 ± 2.78a 24.1 ± 0.47ab 496.6 ± 67.45a

Salt-adapted rice R1 cell line R1A0
One 2 4 5 6 9 10 12 13 14 15 16 17 18 20 21 23 24 25 26 27 R1A60
One 2 3 4 5 6 7 8 9 10 11 13 14 15 17 20 21 22 24 25 26 27 28 31 33 34 35 36 37 38 R1A80
One 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 R1A100
One 2 3 4 5 6 7 8 9 R1A120
One 2 3 4 5 6 7 8 9 10 11 12 13 14

Example 4: Investigation of rice paddy root and yield components

According to one embodiment of the present invention, the A120 line seeds of the R1 seeds in Table 4 were diluted with 2000: 1 oil of prochloraz (25%) in water and disinfected at 25 DEG C for 24 hours. Day. The seeds of 2-3㎜ long leaves and root seeds were transplanted into the soil for general rice and then purified in the greenhouse for 3 weeks. The number of tillers, the number of seeds, the number of seeds and the number of chaffs were counted at the harvest time. The statistical analysis was performed by SAS system using Duncan's multiple range test method at a level of 5% ).

As a result, as shown in Tables 5 and 6, the short plant height, the low ripeness rate and the degree of ripeness shown in the R0 generation regeneration rice showed a phenotype recovering even if not fully developed, The phenotypic phenotypes were also recovering from the R0 generation. The seeds of R1 generation (R2 seed) were obtained after harvesting, and the lines of regenerated rice obtained seeds are shown in Table 7 below.

lines Length (cm) Number of teeth (per m ² ) Per panicle per ear R1A1120-1 74.3 ± 4.07 abcd 320 ± 89.6 a 129 ± 13.8 a R1A1120-2 75.4 ± 6.62 abc 285 ± 73.3 a 128 ± 17.7 a R1A1120-5 68.6 ± 3.20 f 331 ± 60.2 a 96 ± 14.0 c R1A1120-6 67.4 ± 2.99 f 291 ± 62.1 a 99 ± 10.4 c R1A1120-7 72.8 ± 3.33 cd 310 ± 94.7 a 119 ± 16.9 ab R1A1120-8 69.9 ± 4.13 ef 317 ± 64.5 a 100 ± 16.6 c R1A1120-9 72.9 ± 4.28 cd 238 ± 78.6 bc 129 ± 14.3 a R1A1120-10 76.8 ± 5.49 a 193 ± 36.9 c 120 ± 18.2 ab R1A1120-11 73.3 ± 4.46 bcd 210 ± 63.2 bc 118 ± 21.5 ab R1A1120-14 72.1 ± 4.27 232 ± 62.7 bc 114 ± 13.7 b WT 75.9 ± 2.63 ab 241 ± 60.8 b 118 ± 16.4

lines Maturity rate (%) Ginseng (g) Yield (brown rice, kg / 10a) R1A1120-1 89.0 + - 2.22 22.1 ± 0.73 c 805.9 ± 213.73 a R1A1120-2 85.8 ± 3.63 22.3 + 1.42 c 690 ± 173.87 b R1A1120-5 69.9 ± 12.41 b 22.3 + - 0.61 c 488.8 ± 128.89 cd R1A1120-6 73.1 ± 13.48 b 22.0 ± 0.71 c 454.4 ± 112.81 d R1A1120-7 86.1 ± 6.31 a 21.9 ± 0.81 c 712.5 ± 272.96 ab R1A1120-8 74.5 ± 17.92 b 22.2 ± 0.81 c 531.0 ± 195.72 cd R1A1120-9 85.1 ± 4.26 a 22.1 ± 1.16 c 579.5 ± 207.96 c R1A1120-10 85.4 ± 3.76 23.1 ± 1.11 b 449.6 ± 86.74 d R1A1120-11 85.9 ± 3.87 a 22.9 ± 1.06 b 494.1 ± 187.62 cd R1A1120-14 85.6 ± 4.28 a 22.3 ± 0.80 c 498.4 ± 128.34 cd WT 87.3 ± 3.71 a 23.7 ± 0.54 a 578.8 ± 120.12 c

Salt-adapted rice R2 cell line R2A120-1 One 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 R2A120-2 One 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 R2A120-5 One 2 3 4 5 6 7 9 10 11 12 13 14 15 16 17 18 19 R2A120-6 One 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 19 R2A120-7 One 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 R2A120-8 One 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 R2A120-9 One 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 R2A120-10 One 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 R2A120-11 One 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 R2A120-14 One 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Example  5: Selection of varieties

According to one embodiment of the present invention, in order to induce embryogenic callus from rice seeds, the degree of salt resistance in six cultivars such as general cultivated rice, rice, hanami, Dongjin, Respectively.

Specifically, the seeds from which the seed coat was removed with a brown rice ball were disinfected once with 70% ethanol for 10 minutes, and then sterilized twice for 20 minutes by adding Triton x-100 to a 1.2% sodium hypochlorite solution at a ratio of 1000: The seeds were sterilized in MS medium supplemented with 200 mM NaCl, and seeds were seeded in two petridishes, each of 10 seeds, and cultured at 25-26 캜 under long-day conditions (LD 16: 8 h). One week later, the seedlings were counted to determine the germination rate among the cultivars. The seeds sterilized by the same method as that for measuring the germination rate were exposed to MS medium at 25-26 ° C for 1 week under long-day conditions (LD 16: 8h) Were transferred to MS medium supplemented with 200 mM NaCl and cultured for 1 week under the same conditions.

As a result, there was a difference in germination rate from the lowest germination percentage (26%) to 43.3% (Dongjin), and the median value was 34.65%. In addition, the length of the top part was the lowest of 2.3 cm (tall) to 4.1 cm (maximum), and the median value was 3.2 cm. As a result of the above experiment, even though salt tolerance was improved when six varieties were weakly resistant to salt, the improved level was similar to the varieties resistant to salt in existing varieties, The selection of varieties as a middle - level response to salt was possible because it may be close to the limit of improvement in salt tolerance and relative comparison may be difficult.

Example 6: Salt resistance test using R1 seed

According to one embodiment of the present invention, the seeds sterilized in the same manner as the germination rate were placed on LB medium supplemented with 200 mM NaCl and placed in a 25-26 ° C, long-day condition (LD 16: 8h) Germination rates were calculated by counting individuals with leaflets between A0 and A120 cell lines. In addition, proliferating callus was obtained by seed sterilization and cell culture of A120 line and control group of R1 seeds. To determine the improvement of salt resistance, proline, glutamine 0.5g / L, casamino acid 0.3g / L culture medium containing 2 mM NaCl and 2 mM NaCl, and culture medium containing 25 mM NaCl and 100 mM NaCl for 4 weeks in the absence of light. Respectively.

As a result, the germination rate of the control group was 50%, whereas that of the A0 group was 25%. The germination rate of the two lines (Table 3, A120-7, A120-9) And 70%, respectively, which was 15-20% higher than that of the control group, indicating that the salt resistance was improved (FIGS. 4 and 5). As a result of comparison of fresh weight, it was confirmed that the A120 line exhibited a higher live weight than the control group in both salt-free conditions and 100 mM NaCl (FIGS. 6 and 7). This suggests that the resistance to salt in the early salt adaptive callus is maintained even after the first generation has developed after the regeneration process, suggesting the possibility of the welfare genetics.

Example 7: R1 Rice Chlorophyll content and ion content measurement

According to one embodiment of the present invention, chlorophyll content and ion content of R1 rice were measured. Specifically, A120 line of R1 seed and control seed (rice) seeds were diluted with water (2000: 1) of proton (prochloraz 25%) and treated for 24 hours at 25 ℃ without light for 24 hours. The seeds were grown at room temperature, and the seedlings having a length of about 2-3 mm and the seeds with roots were transferred to general water and cultured in a hydroponic culture system for 2 weeks in a chamber of 25 ° C, 55-60% relative humidity and long day (LD 16: 8h) . Then, the medium was exchanged with 1/5 MS (no sucrose) liquid medium and cultured for 1 week. The seedlings of the third leaf stage (weaning period) of the 3 weeks-old plants were transferred to a glass test tube having a diameter of 3 cm and a depth of 20 cm, Chlorophyll content and ion content were measured in 1/5 MS (no sucrose) liquid medium supplemented with NaCl for 1 week with salt stress. Chlorophyll content was measured by extracting the second leaf from each of 3 independent individuals of R1 A120 and 1 ml of DMSO per 50 mg of live weight. The chlorophyll was extracted for 2 days at 30 ℃ under no light condition. The chlorophyll content extracted using a spectrophotometer was measured using the following formula.

Total chlorophyll content (㎍.mg-1fw) = [{(20.29 × A645) + (8.02 × A663)} × DMSO ml] / fresh weight mg

Further, the ion content measured is above-ground and after 2 days drying at 60 ℃ dividing the underground part nitric acid _{(HNO 3 70%) ICP-} MS (inductively coupled plasma-mass spectroscopy) to pre-treatment into a 5 ml K ^+, Na ^+, and the Ca ^{2 +} ions were measured.

As a result, the chlorophyll content in R1 A120 was higher than that in the control group (Fig. 8). The ion content of K + ions in the ground / underground portion and Na ⁺ ions in the ground portion Was higher than that of the control group and lower than that of the control group. It is notable that, in general, when a plant is exposed to salt stress, the Na ⁺ ion absorbed from the root (underside) interferes with the absorption of ions such as K ⁺ and Ca ^{2 +} , and moves to the upper part simultaneously with the osmotic disorder to inhibit photosynthesis and respiration causing sikineunde cause plant growth disorders, 100mM NaCl treatment during while the Na ± ions in a large amount in the absorbent as long as the aerial Na ⁺ ions are detected by the below-ground of the control group R1 A120 is also the absorbed Na ⁺ ion quantity in the below-ground plenty than in the control group However, it was confirmed that a smaller amount was detected in the upper part than in the control (Figs. 9 to 11). In addition, regardless of the salt conditions, the K ⁺ / Na ⁺ ratio was higher in R1 A120 than in the control (Fig. 12), indicating that the physiological characteristics of regenerated rice in high salt (120 mM NaCl) / radish relationship without K ⁺ ion absorption mechanism is Na ⁺ ions to generate a higher level than the existing and the mechanism of high salt (100 mM NaCl) conditions, isolate the Na ⁺ ion absorption from the roots or motion restraint, such as occurs in the above-ground is Suggesting that it contributes to improved salt resistance by alleviating aggregation.

Example 8: R1 rice phenotype survey

For the phenotypic investigation, phenotypic analysis of the salt in the greenhouse maintained at a temperature of 23-27 ° C was carried out on rice line R1 (ROA0, ROA60, ROA80, ROA100, ROA120) according to an embodiment of the present invention. Specifically, the seeds were diluted with water (2000: 1) of prochloraz (25%), diluted in water, sterilized at 25 ° C for 24 hours and then seeded in flowing water for 3-4 days at room temperature. After the transplantation of rice seeds from the rice field, four weeks were placed in the greenhouse. After this, 100 mM NaCl was treated and the phenotype was examined at 4th and 8th weeks.

As a result, the regenerated rice with higher salt adaptation concentration tended to have a shorter plant height and higher tillering. In the treated group, there was no significant difference in plant height between the two groups. And it was confirmed that the rate of dryness of leaf ends was relatively low (FIG. 13). The tendency of the number of tillers shown in the control group was similar to the number of tillers among the number of the components of the rice plants regulated in the general condition, And the possibility of welfare genetics in which a similar phenotype is maintained.

Example 9 R2 Rice Salt Resistance Validation Screening

According to one embodiment of the present invention, R2 Rice Salt Resistance Screening was performed by diluting R 2 A120 line and control (rice) rice seeds with 2000: 1 crude oil of prochloraz (25%), After 24 hours of disinfection, it was washed in running water for 5 minutes and then dipped in clean water for 10 minutes. After washing for 5 minutes, the seeds were sprouted with a wiper (kimtech-wiper) moistened with a third-order distilled water in a petri dish having a diameter of 9-10 cm, and germinated for 3 days at 30 ° C in the absence of light. For the A120 line, which is 3-4cm in length and 3-4cm in length, 50 rice seedlings of 1-3cm length were transferred and treated with water containing 50 mM NaCl at 24-25 ℃, Humidity 55-60%, long-day condition (LD 16: 8h). Water containing 50 mM NaCl was added so that the seed coat with the endosperm was not dried, and the whole was exchanged with fresh 50 mM NaCl water every one week. Survival rates of rice seedlings were screened up to 22 days at intervals of 2 days from day 12 of growth. On the other hand, the irradiation method was classified into 5 stages as shown in Table 8 below, and it was judged that the rice in steps 1 and 2 survived.

As a result, the survival rate of the control group was 31.2 ± 8.2% based on the 16th day after treatment with 50 mM NaCl on day 12, and 19 out of 42 lines of R2 A120 line showed higher survival rate than the control group. Of the 19 lines with higher survival rates than the control group, 10 lines showed a survival rate of more than 50%, but they belong to the first and second stages of Table 8 below. However, the lines with low chlorophyll content, Six lines were excluded (Fig. 14). The results showed that the salt resistance obtained by regeneration after adaptation of callus to organic salt at high salt (120 mM NaCl) condition in general rice (Japanese rice) was cultivated in general package without salinity and progressed two generations This suggests that the acquired high adaptability is memory (generation stress memory phenomenon) even after the generation progress.

step Phenotype Resistance One Normal growth; No symptoms of leaves High resistance 2 Almost normal growth; Chlorosis or rolled leaf tip Resistance 3 Some inhibition of growth; Most of the leaves are curled; Some leaves only grow (elongation) usually 4 Growth arrest; Mostly leaf blight; Some plants dying sensibility 5 Most objects die or die High sensitivity

In conclusion, the method of increasing the salt tolerance of a plant of the present invention provides a method of enhancing stress tolerance of a plant without introducing homologous or heterologous genes using transgenic plants, thereby increasing salt tolerance compared to wild-type plants, It is also effective in the development of plants that can grow strongly in the affected area and in the increase of the productivity of crops, and also in the development of transgenic crops for production of high value-added protein fish oral vaccines or industrial enzymes derived from plants.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

Claims (6)

An organic step of disinfecting and washing the rice seeds and then allowing the seeds to stand in a medium at a temperature of 24 to 27 ° C and in a light-free condition for 5 to 6 weeks; And
Wherein the callus selected in said organic step is cultured at a temperature of 24-27 DEG C and in the absence of light in a stepwise increase in the concentration of the salt. Increased salt tolerance of rice. The method according to claim 1,
The method of claim 1, wherein the seeds are seedless. The method according to claim 1,
Wherein the salt is NaCl. The method according to claim 1,
Wherein the concentration of the salt in the culturing step is increased stepwise from 110 to 130 mM at 10 to 30 mM. 5. The method of claim 4,
Wherein the salt concentration is increased stepwise by 10 to 30 mM in stepwise manner. The method according to claim 1,
Wherein the culturing step is performed for each step for 3 to 5 weeks.

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