KR20170072747A - Method for hydroponics of nicotina benthamiana - Google Patents
Method for hydroponics of nicotina benthamiana Download PDFInfo
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- KR20170072747A KR20170072747A KR1020150181308A KR20150181308A KR20170072747A KR 20170072747 A KR20170072747 A KR 20170072747A KR 1020150181308 A KR1020150181308 A KR 1020150181308A KR 20150181308 A KR20150181308 A KR 20150181308A KR 20170072747 A KR20170072747 A KR 20170072747A
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- tobacco
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- hydroponic cultivation
- plant
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G31/00—Soilless cultivation, e.g. hydroponics
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- A01G1/001—
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
Abstract
The present invention relates to a hydroponic method in tobacco plants, more specifically to tobacco plants (Nicotiana The present invention relates to a hydroponic cultivation method of a tobacco plant including a step of hydroponically cultivating seedlings of benthamiana . According to the present invention, when tobacco plants are hydroponically cultivated in a closed plant production system, the environmentally friendly production and growth promotion of tobacco plants can be obtained. In addition, when the cultivation method of tobacco plants according to the present invention is applied to a transgenic tobacco plant, it is possible to efficiently produce an object such as a high-quality pig swine antigen vaccine raw material.
Description
The present invention relates to a hydroponic cultivation method of a tobacco plant, and more particularly, to a hydroponic cultivation method capable of promoting the growth of a tobacco plant by supplying a growing environment and an appropriate nutrient solution in a closed plant production system.
1-year-old herbaceous plant person Tobacco plants are branches and tobacco plants and are native to Australia. Tobacco is a botanical source of tobaccoNicotina tabacum), And the maximum length is 1.5m and the width does not exceed 0.2m. Generally, tobacco plants are tobacco (Nicotina tabacumUnlike It is not cultivated for commercial use, and is used for research in universities and research institutes.
Tobacco plants are representative unicellular plants, and flowering occurs at a limit below the limit. In other words, when the length of night is lengthened, flower bud differentiation is induced. In case of transgenic tobacco plants, if the flowering is carried out, pollen may be released, so it must be cultivated under the closed system so that it can be safely produced without ecological disturbance.
Therefore, in a general research facility, tobacco plants are grown using a greenhouse or plant growth phase, in which the cultivation method is in the form of two cultivars with soil cultivation using pots. On the other hand, the transgenic plants confirmed and verified at the laboratory level are subjected to revalidation and economical analysis through mass production. In this case, mass production requires a higher yield per unit area than the soil cultivation, shortening the growth period, Which makes it easy to manage the nutrient solution. In addition, hydroponics can be cultivated with only about 10% of the source of the cultivated land, and there is no loss of fertilizer (Burrage, SW 1992. NFT in protected cultivation Acta Hort 323: 25-38).
Thus, as described in Korean Patent Laid-Open Publication No. 10-2014-0045111, a hydroponic cultivation method using a culture medium is partially introduced into a special crop, but research on hydroponic cultivation methods for transgenic plants is insufficient.
On the other hand, swine fever virus is an infectious disease controlled by a livestock and has a high prevalence rate and high mortality rate during infection. When a pig fever occurs, it is a major obstacle to the stoppage of pork export as well as direct damage such as our company. Domestic use of a highly effective Ro-Vaccine (live vaccine) as a countermeasure against swine fever in Korea is problematic because it has a disadvantage in that it can not distinguish between vaccination and outbreak of infection. Recently, as described in Korean Patent Application No. 10-2014-0185686, the development of a transgenic plant for producing a marker vaccine capable of distinguishing between the white stretch and the outdoor infection axis has been completed, and the thus transformed tobacco plant Nicotiana benthamiana ), and the efficacy of the target animal was verified with the vaccine.
Therefore, when a hydroponic cultivation technique that can be applied to a closed plant production system and efficiently cultivate a tobacco plant is developed, it can be widely applied in the related field including the efficient production of the raw material for the pig fever antigen vaccine .
Accordingly, one aspect of the present invention provides a method for hydroponics of tobacco plants.
According to one aspect of the present invention, tobacco plantsNicotiana benthamiana) Of a tobacco plant is hydroponically cultivated.
The tobacco plant is preferably a transgenic tobacco plant for the production of swine fever antigen vaccine.
It is preferable that the hydroponic cultivation is carried out by a circulating-saline solution hydroponic culture (DFT) or a circulating thin-film hydroponic culture (NFT) method.
The temperature during hydroponic cultivation is preferably 20.5 to 32.5 ° C.
It is preferable that the light period during the hydroponic cultivation is 14 to 18 hours.
It is preferable that the aforementioned range is irradiated with light with a light intensity of 120 to 300 μmol / m 2 / s.
The relative humidity in hydroponic cultivation is preferably 55 to 65%.
The hydroponic cultivation period is preferably 7 days to 28 days.
The seedling of the tobacco plant is preferably obtained by a seedling step carried out in a medium consisting of a mixture of commercial horticultural soil, commercial soil and vermiculite in a volume ratio of 1: 1 to 3: 1, a rock surface and a combination thereof .
The medium is preferably a medium on which a commercial soil is packed on a rock surface.
The period of time during which the seedling stage is carried out is preferably 15 to 20 days.
According to the present invention, when tobacco plants are hydroponically cultivated in a closed plant production system, the environmentally friendly production and growth promotion of tobacco plants can be obtained. In addition, when the cultivation method of tobacco plants according to the present invention is applied to a transgenic tobacco plant, it is possible to efficiently produce a target such as a high-quality pig swine fever antigen vaccine raw material.
FIG. 1 is a graph showing the results of the seeding rates of the transgenic tobacco seeds according to the type of seeds.
Fig. 2 is a graph showing the results of living body weight of the transgenic tobacco cultured at the time of nursing.
FIG. 3 is a graph showing the results of living body weight of the transgenic tobacco according to the number of seedling growing days.
FIG. 4 is a graph showing the results of live body weight of each transplanting tobacco culturing method. FIG.
FIG. 5 is a graph showing changes in the net photosynthetic rate by the light intensity (a) and the temperature (b) during the cultivation of the transgenic tobacco according to an embodiment of the present invention.
FIG. 6 is a graph showing the results of the shoot FW (a) and the content (concentration) of swine fever antigen protein in the cultivation period according to the cultivation period in the cultivation of the transgenic tobacco according to an embodiment of the present invention.
FIG. 7 is a graph showing the results of the growth of the nutrient solution and the biomass of the swine fever antigen protein in hydroponic cultivation of the transgenic tobacco according to an embodiment of the present invention.
Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings. However, the embodiments of the present invention can be modified into various other forms, and the scope of the present invention is not limited to the embodiments described below.
The term " closed plant production system "as used herein means a plant and equipment for continuously producing plants throughout the year regardless of the external climate by artificially controlling the growth environment of light, temperature, humidity and nutrient solution in a closed facility.
"Raising seedlings " described in this specification refers to the seeding of seeds for the production of crops to produce juvenile plants.
The term " recirculating saline solution type "described herein is a system in which plants sown with an artificial medium are grown in a culture medium prepared from raw water and water-soluble nutrients. The culture medium is maintained at a constant height in a water tank for a predetermined time, And the like.
The "recirculating thin-film hydroponic system" described in the present specification is a system in which a culture medium is continuously flowed in a cultivating zone having a certain gradient when a cultivated plant is planted, and the roots of the crop absorb the culture medium, And so on.
The term " planting " as used herein means planting at a place where light, temperature and humidity are controlled after planting, and planting it at a place to be cultivated after a certain period of time.
According to the present invention, there is provided a hydroponic cultivation method capable of promoting the growth of tobacco plants. More specifically, the present invention relates to tobacco plants ( Nicotiana benthamiana ) seedlings.
In this specification, the Nicotiana benthamiana is distinguished from Nicotina tabacum and is called "tobacco plant" and "tobacco", respectively.
The tobacco plant of the present invention may be a transgenic tobacco plant for producing a swine fever antigen vaccine and may be applied to a transgenic tobacco plant for producing a swine fever antigen vaccine to promote the growth of tobacco plants by applying the hydroponic cultivation method of the present invention The target antigen vaccine can be efficiently produced.
The germination stage for obtaining the seedlings to which the hydroponic cultivation method of the present invention is applied is not particularly limited, but it is preferable that the light-shade period is 12 h / 12 h and the light intensity is 120 to 300 μmol / m 2 / s. < / RTI > On the other hand, the planting of the tobacco plant can be carried out on a catching rock surface cell, and the soil can be carried out as a commercial soil for general horticulture. That is, the hydroponic cultivation method of the present invention may be carried out on seedlings obtained by germinating tobacco plant seeds or commercially obtained seedlings germinated.
The hydroponic cultivation of the present invention can be carried out by a circulating fluidized-bed dipping (DFT) or a circulating thin-film hydroponic cultivation (NFT) method, preferably by a circulating thin-film hydroponic culture (NFT) method.
The light period during the hydroponic cultivation is 14 to 18 hours, preferably 14 to 17 hours, more preferably about 16 hours. If the specified period in the light period is less than 14 hours There is a problem that the flowering is induced early, and when it exceeds 18 hours, there is no difference in growth There is an uneconomical problem in terms of power consumption. For example, the light irradiation may be performed at a light intensity of 16 h / 8 h during the growing and growing period.
On the other hand, in the light and dark cycle, the light is preferably irradiated at a light intensity of 120 to 300 μmol / m 2 / s, more preferably 170 to 200 μmol / m 2 / s. When the light irradiation is performed at a light intensity of less than 120 μmol / m 2 / s, there is a problem that the net photosynthetic rate is inadequate. When the light irradiation is performed at a light intensity exceeding 300 μmol / m 2 / s, The degree of increase in the net photosynthetic rate is not so large and tends to be uneconomical.
The light intensity is based on the light intensity measured at a distance of about 20 cm from the light source.
In the present invention, the light irradiation may use fluorescent lamps or light emitting diode elements, but the present invention is not limited thereto, and the artificial lighting may be performed during all the nursery and cultivation period after sowing of tobacco plants.
The temperature during hydroponic cultivation is 20.5 to 32.5 ° C, preferably 23 to 27 ° C. There is a problem that the net photosynthetic rate is lowered when the temperature in the hydroponic cultivation is less than 20.5 ° C or exceeds 32.5 ° C.
When the relative humidity is lower than 55%, there is a problem of loss of the nutrient solution supplied. When the relative humidity is higher than 65%, the problem of excessive humidity and fungal disease occurs have.
The hydroponic cultivation of the tobacco plant of the present invention can be carried out in a closed type constant temperature and humidity chamber which can maintain a preferable temperature and humidity range as described above.
In the present invention, the hydroponic cultivation period is preferably 7 days to 28 days. More specifically, the protein content (concentration) on the 7th day and the 14th day after the planting was relatively high, and the protein content (concentration) decreased on the 21st day and the 28th day after the lapse of the period. Day. Therefore, it is preferable that the cultivation period is 7 to 28 days. The protein content tends to be inadequate when the hydroponic cultivation period is less than 7 days, and the protein content tends to be lower when the hydroponic cultivation period exceeds 28 days.
On the other hand, the seedling of the tobacco plant to which the hydroponic cultivation method of the present invention is applied is a medium in which a commercial soil for horticulture, a commercial soot and a commercial soil and a vermiculite are mixed in a volume ratio of 1: 1 to 3: It is preferable that the medium is obtained by a seedling step to be performed, more preferably, the medium is a medium in which a commercial soil is coated on a rock surface.
At this time, the period for carrying out the seedling stage is preferably 15 days to 20 days, and when the seedling growing time is less than 15 days, there is a problem that the root can not be settled. When it exceeds 20 days, there is a problem of aging of the seedling.
The culture solution that can be used in the present invention is not particularly limited, but it can be used as a preparation for Otsuka paprika culture solution or Yamazaki lettuce culture solution.
According to the present invention, when tobacco plants are hydroponically cultivated in a closed plant production system, the environmentally friendly production and growth promotion of tobacco plants can be obtained. In addition, when the cultivation method of tobacco plants according to the present invention is applied to a transgenic tobacco plant, it is possible to efficiently produce an object such as a high-quality pig swine antigen vaccine raw material.
Hereinafter, the present invention will be described more specifically by way of specific examples. The following examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited thereto.
Example
1. Acquisition of tobacco plants
As a target plant for applying the hydroponic cultivation method of the present invention, a seed of a transgenic tobacco plant (Nicotiana benthamiana) for producing a pig fever antigen vaccine was obtained from Bio-App, seeded in a catching rock surface cell, The seeds were germinated at a temperature of 28 to 30 캜. At this time, the contrast period was 12 h / 12 h, and the light intensity was maintained at 120 to 300 μmol / m 2 / s.
2. Hair growth method experiment of tobacco plant
In order to produce seedlings of the transgenic tobacco obtained in the above 1 under the closed plant production system of the present invention, experiments were conducted with different types of seedlings and seedlings.
(1) Badge By type Maternity rate And Raw weight Confirmation experiment
The culture medium was constructed as follows using a commercial soil for gardening (CS), a vermiculite (VL) grow foam (GF), a sponge (S) and a rock surface (RW).
1) Commercial soil for gardening (CS)
2) A mixture of soil and vermiculite (VL) so that the volume ratio of vermiculite (VL) is 3: 1
3) A mixture of soil and vermiculite (VL) so that the volume ratio of vermiculite (VL) is 1: 1
4) A mixture of soil and vermiculite (VL) so that the volume ratio of vermiculite (VL) is 1: 3
5) Vermiculite (VL)
6) Glow form (GF)
7) Sponge (S)
8) Rock surface (RW)
The seeding rate of the transgenic tobacco plants obtained in 1) above was confirmed on the 14th day after sowing, and as a result, as shown in Fig. 1, 85% or more of 1), 2), 3), 6) and 8) High. On the other hand, in the vermiculite medium and the sponge (S), the nap rate was remarkably low.
On the other hand, as a result of measuring the weight of seedlings at 14 days after sowing of the transformed tobacco plants obtained in above 1., as shown in Fig. 2, And the lowest in the treatment with vermiculite medium of 5).
As a result, it can be confirmed that it is most preferable to cover the rock surface with the rock surface during nursing of the transgenic tobacco.
(2) According to the seedling period Raw weight Confirmation experiment
As shown in 2. (1) above, the fresh weight of the transgenic tobacco seedlings of the first seedling period was measured after covering with the soil of the horticultural field in the rock-bottom culture medium.
As a result, as can be seen from FIG. 3, the body weight was significantly increased from
3. Cultivation
By method
Experiment for confirmation of growth change
The tobacco plants were fed with two correlated water samples (CS), and circulating type DFT and NFT were performed to test the changes in the growth of the tobacco plants by the irrigation method. Respectively.
As shown in FIG. 4, the fresh weight of the ground portion was the highest at about 70.5 g in the cyclic thin film hydroponic system (NFT) As a result, it was confirmed that the cultivation of transgenic tobacco was effective for the hydroponic cultivation of transgenic tobacco using the circulating thin film hydroponic system (NFT).
4. Experiment to confirm the change of growth according to hydroponic cultivation condition
Experiments were carried out to investigate the growth of tobacco plants and the contents of pig fever antigen protein by cultivating period, temperature and light treatment, and culture media for hydroponic cultivation. In this experiment, a cyclic thin film hydroponic system (NFT) was applied.
(1) Temperature and luminous intensity At the net photosynthetic rate Impact confirmation experiment
The effects of temperature and light intensity on the net photosynthetic rate of transgenic tobacco plants were investigated during the cyclic thin - film hydroponic cultivation.
As a result, as shown in FIG. 5 (b), the net photosynthetic rate was remarkably high at a temperature of about 25 ° C., and the net photosynthetic rate at a light intensity of 120 to 200 μmol / m 2 / s And it increased rapidly.
Therefore, considering the net photosynthetic efficiency of circulating thin film hydroponic culture of transgenic tobacco, it is desirable that the temperature is about 25 ° C and that the light intensity of the artificial light source is at least 120 μmol / m 2 / s when measured at a distance of 20 cm from the light source .
(2) Cultivation By period Growth experiment
Growth of the transgenic tobacco plant was investigated during the cultivation period.
As a result, as shown in Fig. 6 (a), the fresh weight rapidly increased until 28 days, and the fresh weight of the upper part was 128 g on the 28th day. On the other hand, as a result of quantitative analysis of the antigen protein, the protein content in the transformed tobacco was relatively high at 7 and 14 days after the formalinization as shown in Fig. 6 (b) It was confirmed that the protein content was lowered.
However, in consideration of the tendency to increase the weight of the ground part in Fig. 6 (a), it can be deduced that the cultivation period is desirable to be cultivated until the 28th day after the planting.
(3) Nutrient solution Growth experiment by composition
Growth experiments of the nutrient solution were carried out during the cyclic thin - film hydroponic cultivation after the formalization of the transgenic tobacco plants.
The culture medium used herein was as shown in Table 1 below and was used in the form of Ross, Soneveld, Yamazaki and Otsuka, respectively. The concentration of each culture was 1.2 ds / m .
As shown in FIG. 7 (a), the highest growth was observed in the Yamazaki and Oatsuka cultures, and the difference in the concentration of antigen protein between the cultures was not significant (b).
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 exemplary embodiments, but, on the contrary, It will be obvious to those of ordinary skill in the art.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109042208A (en) * | 2018-08-24 | 2018-12-21 | 湖北省农业科学院植保土肥研究所 | A kind of cultural method promoting root system development tobacco growth early period |
| KR20210050814A (en) | 2019-10-29 | 2021-05-10 | 강동국 | Capsule for controlling concentration of nutrient solution and use of the same |
Citations (4)
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| KR20030096842A (en) * | 2002-06-18 | 2003-12-31 | 홍학표 | Simple agricultural plot using culture ground methods for home use |
| KR101105311B1 (en) * | 2011-06-09 | 2012-01-18 | 이용우 | Appratus for cultivating plant |
| KR20140104707A (en) * | 2013-02-21 | 2014-08-29 | 박아론 | Antigen Producing Apparatus for Vaccine and Method thereof Using Plant |
| KR20150018568A (en) * | 2012-06-13 | 2015-02-23 | 엥거니 제네틱스 | Method for Producing High-quality Recombinant Allergens in a Plant |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20030096842A (en) * | 2002-06-18 | 2003-12-31 | 홍학표 | Simple agricultural plot using culture ground methods for home use |
| KR101105311B1 (en) * | 2011-06-09 | 2012-01-18 | 이용우 | Appratus for cultivating plant |
| KR20150018568A (en) * | 2012-06-13 | 2015-02-23 | 엥거니 제네틱스 | Method for Producing High-quality Recombinant Allergens in a Plant |
| KR20140104707A (en) * | 2013-02-21 | 2014-08-29 | 박아론 | Antigen Producing Apparatus for Vaccine and Method thereof Using Plant |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109042208A (en) * | 2018-08-24 | 2018-12-21 | 湖北省农业科学院植保土肥研究所 | A kind of cultural method promoting root system development tobacco growth early period |
| KR20210050814A (en) | 2019-10-29 | 2021-05-10 | 강동국 | Capsule for controlling concentration of nutrient solution and use of the same |
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