KR102194910B1 - Cultivation method of in-vitro-cultured plantlet of tea plant with increased root growth by control of light quality - Google Patents
Cultivation method of in-vitro-cultured plantlet of tea plant with increased root growth by control of light quality Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
- A01H5/06—Roots
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- F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
- F21—LIGHTING
- F21W—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES F21K, F21L, F21S and F21V, RELATING TO USES OR APPLICATIONS OF LIGHTING DEVICES OR SYSTEMS
- F21W2131/00—Use or application of lighting devices or systems not provided for in codes F21W2102/00-F21W2121/00
- F21W2131/10—Outdoor lighting
- F21W2131/109—Outdoor lighting of gardens
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- F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
- F21—LIGHTING
- F21Y—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES F21K, F21L, F21S and F21V, RELATING TO THE FORM OR THE KIND OF THE LIGHT SOURCES OR OF THE COLOUR OF THE LIGHT EMITTED
- F21Y2113/00—Combination of light sources
- F21Y2113/10—Combination of light sources of different colours
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- F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
- F21—LIGHTING
- F21Y—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES F21K, F21L, F21S and F21V, RELATING TO THE FORM OR THE KIND OF THE LIGHT SOURCES OR OF THE COLOUR OF THE LIGHT EMITTED
- F21Y2115/00—Light-generating elements of semiconductor light sources
- F21Y2115/10—Light-emitting diodes [LED]
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Abstract
Description
본 발명은 광질 조절에 의한 뿌리 생육이 증진된 차나무 기내배양묘의 배양방법에 관한 것으로, 보다 상세하게는 증식배양 단계에서 적색광, 청색광 및 백색광이 동일한 광량비율로 혼합된 LED 인공광원을 차나무 유묘에 조사하여 생육이 향상된 차나무(Camellia sinensis L.) 기내배양묘를 생산하는 방법에 관한 것이다.The present invention relates to a method of culturing tea tree seedlings with enhanced root growth by light quality control, and more particularly, irradiating an LED artificial light source in which red light, blue light and white light are mixed at the same light quantity ratio in the growth culture step to the tea tree seedlings. Thus, it relates to a method of producing in-flight cultured tea tree ( Camellia sinensis L.) with improved growth.
차나무(Camellia sinensis L.)는 동백나무과에 속하는 아열대성 상록의 다년생 종자식물로 열대지방에서 온대지방에 이르기까지 광범위하게 분포, 재배 및 생산되고 있다. 차나무는 잎을 이용하여 다양한 형태의 차를 만들어 음용하는 기호 음료류의 식물로 약 3,000여 년 전부터 우리생활에 함께 해온 것으로 알려졌다. 최근에는 건강에 대한 국민적인 관심 그리고 건강기능성에 대한 차 효능의 증명, 전통 다도의 재등장으로 차의 수요가 많아질 것으로 예상됨에 따라 차나무 재배가 가능한 지역에서는 경쟁적으로 차 재배면적을 확대하고 있다.Tea tree ( Camellia sinensis L.) is a subtropical evergreen perennial seed plant belonging to the Camellia family and is widely distributed, cultivated and produced from tropical to temperate regions. Tea trees are known to have been with us for about 3,000 years as a favorite beverage plant that makes various forms of tea using leaves and drinks it. In recent years, as the demand for tea is expected to increase due to the public interest in health, proof of the efficacy of tea for health functions, and the re-emergence of traditional tea ceremony, the area where tea trees can be cultivated is competitively expanding the tea cultivation area.
차나무는 영양번식과 실생번식을 하는 타가수정 식물로, 국내 다원(茶園)의 일부 기업적 재배지를 제외하고는 대부분이 주로 실생번식하는 실정이다. 식물 조직배양기술은 식물체의 무병주 생산, 반수체 및 순계육성, 유전자 보존 등에 기여하고 있으며, 특히 단기간 내 대량증식을 위한 기내 미세증식법(micropropagation)이 가장 많이 연구되고 있다. 지금까지 차나무의 기내배양묘 생산과 관련된 국내 연구는 주로 대량증식을 위한 생장호르몬제 처리 및 적정배지 선발 위주로 이루어 졌다.The tea tree is a taga fertilization plant that reproduces vegetatively and real life, and most of the tea trees, except for some corporate cultivation areas in domestic tea gardens, are mainly real-life propagation. Plant tissue culture technology contributes to the disease-free production of plants, haploid and pure breeding, and gene preservation, and in particular, micropropagation for mass propagation within a short period of time is being studied the most. Until now, domestic studies related to the production of in-flight cultured tea tree have mainly focused on treatment with growth hormones for mass growth and selection of appropriate media.
광질은 식물의 생장, 형태, 형성, 색소형성 등에 대한 에너지원 뿐만 아니라 조절인자로 작용하며, 피토크롬(phytochrome)이나 크립토크롬(cryptochrome)과 같은 광수용체의 작용과 관련되어 주로 줄기 신장이나 측지 발생, 엽면적 확대, 화경신장 및 색소 형성 등 기내 및 기외식물체의 광합성, 생장 및 형태형성에 영향을 미치는 것으로 알려져 있다.Mineral quality is not only an energy source for plant growth, morphology, formation, and pigment formation, but also acts as a regulator, and is mainly related to the action of photoreceptors such as phytochrome or cryptochrome, and thus stem elongation or geodetic development, It is known to affect photosynthesis, growth and morphogenesis of in-flight and extra-flight plants, such as leaf area expansion, flower growth, and pigment formation.
한편, 한국공개특허 제2015-0076917호에는 '기내배양에 의한 차나무 대량생산 방법'이 개시되어 있고, 한국등록특허 제1438393호에는 'LED 광원을 이용한 꽃송이 버섯의 균사 배양 방법'이 개시되어 있으나, 본 발명의 '광질 조절에 의한 뿌리 생육이 증진된 차나무 기내배양묘의 배양방법'에 대해서는 개시된 바가 없다.On the other hand, Korean Patent Publication No. 2015-0076917 discloses a'mass production method of tea tree by in-flight culture', and Korean Patent No. 14 38393 discloses a'method for culturing mycelium of blossoms mushrooms using an LED light source'. There is no disclosure of the'cultivation method of in-flight tea tree seedlings with enhanced root growth by light quality control' of the present invention.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자들은 기내(in vitro)에서 발근하여 순화 생존율이 높은 무병의 차나무 기내배양묘를 생산하기 위해서, 기내배양 과정 중 증식 단계에서 차나무 유묘에 적색광, 청색광 또는 혼합광(적색광:청색광:백색광, 1:1:1)의 LED 인공광원을 조사하며 배양한 결과, 혼합광 처리구에서 차나무 기내배양묘의 뿌리 생육 특성이 대조구(형광등) 및 다른 인공광원 처리구에 비해 우수한 것을 확인함으로써, 본 발명을 완성하였다.The present invention was derived from the above requirements, in order to produce disease-free tea tree seedlings with high acclimatization survival rate by rooting in vitro , red light on tea tree seedlings during the growth stage during in-flight culture process. , Blue light or mixed light (red light: blue light: white light, 1:1:1) LED artificial light source was irradiated and cultivated. As a result, the root growth characteristics of the in-flight cultured seedlings of tea trees in the mixed light treatment group were controlled by the control (fluorescent lamp) and other artificial light source treatment tools. By confirming that it is superior to that, the present invention was completed.
상기 과제를 해결하기 위해, 본 발명은 기내(in vitro)에서, 차나무 유묘에 LED(Light-emitting diode) 인공광원을 조사(irradiation)하며 배양하는 단계를 포함하는, 뿌리 생육이 증진된 차나무(Camellia sinensis L.) 기내배양묘의 재배방법을 제공한다.In order to solve the above problems, the present invention is on board (in vitro) In the irradiation a LED (Light-emitting diode) an artificial light source to the tea plant seedlings (irradiation), and the two, root growth, comprising the step of culturing promote tea (Camellia sinensis L.) Provides a cultivation method for in-flight cultured seedlings.
또한, 본 발명은 상기 방법에 의해 재배된 뿌리 생육이 증진된 차나무 기내배양묘를 제공한다.In addition, the present invention provides a tea tree in-flight cultivation seedling with enhanced root growth grown by the above method.
본 발명의 광질 조절에 따른 뿌리 생육이 향상된 차나무(Camellia sinensis L.) 기내배양묘 배양방법은 기내에서 발근하여 순화 생존율이 높은 차나무 무병주의 대량증식 및 우량묘 생산 등에 유용하게 활용될 수 있을 것이다.The method of culturing the in-flight cultured tea tree ( Camellia sinensis L.) with improved root growth according to the light quality control of the present invention may be usefully used for mass propagation and production of excellent seedlings of tea tree disease-free stocks having a high acclimatization survival rate by rooting in the plane.
도 1은 다양한 LED 인공광원 하에서 배양된 차나무 기내배양묘의 엽록소 함량을 분석한 그래프이다.
도 2는 다양한 LED 인공광원 하에서 배양된 차나무 기내배양묘의 엽록소 형광이미지(CFI)이다. A: 대조구(형광등), B: 적색광 처리구, C: 청색광 처리구, D: 혼합광 처리구.
도 3은 다양한 LED 인공광원 하에서 배양된 차나무 기내배양묘의 최대양자수율(Fv/Fm) 및 비광화학 소멸(NPQ)을 분석한 그래프이다. y축은 백분율을 의미한다.1 is a graph analyzing the chlorophyll content of in-flight cultured tea tree seedlings cultured under various LED artificial light sources.
2 is a chlorophyll fluorescence image (CFI) of in-flight cultured tea tree seedlings cultured under various LED artificial light sources. A: control (fluorescent lamp), B: red light treatment, C: blue light treatment, D: mixed light treatment.
Figure 3 is a graph analyzing the maximum quantum yield (F v / F m ) and non-photochemical extinction (NPQ) of the in-flight cultured seedlings of tea trees cultivated under various LED artificial light sources. The y-axis means percentage.
본 발명의 목적을 달성하기 위하여, 본 발명은 기내(in vitro)에서, 차나무 유묘에 LED(Light-emitting diode) 인공광원을 조사(irradiation)하며 배양하는 단계를 포함하는, 뿌리 생육이 증진된 차나무(Camellia sinensis L.) 기내배양묘의 재배방법을 제공한다.In order to achieve the object of the present invention, the present invention is a tea tree with enhanced root growth, comprising the step of irradiating and culturing a tea tree seedling with a light-emitting diode (LED) artificial light source in vitro . ( Camellia sinensis L.) It provides the cultivation method of in-flight cultured seedlings.
본 발명에 따른 차나무 기내배양묘의 재배방법에 있어서, 상기 LED 인공광원은 적색광 : 청색광 : 백색광이 1 : 1 : 1의 광량비율로 혼합되어 이루어진 것일 수 있으나, 이에 제한되지 않는다. 또한, 상기 인공광원에 사용된 적색광은 620~640 nm 파장의 광원일 수 있고, 청색광은 450~470 nm 파장의 광원일 수 있으나, 이에 한정되는 것은 아니다.In the method of cultivating a tea tree in-flight cultured seedling according to the present invention, the LED artificial light source may be formed by mixing red light: blue light: white light at a light amount ratio of 1:1, but is not limited thereto. In addition, the red light used for the artificial light source may be a light source having a wavelength of 620 to 640 nm, and the blue light may be a light source having a wavelength of 450 to 470 nm, but is not limited thereto.
본 발명의 용어 '조사(irradiation)'는 조명 등을 이용하여 인공적으로 빛에 노출시키는 것을 의미하며, '광조사(lighting)'와 혼용되어 사용될 수 있다.The term "irradiation" of the present invention means artificially exposed to light using lighting, etc., and may be used interchangeably with "lighting".
본 발명의 일 구현 예에 따른 차나무 기내배양묘의 재배방법은, 구체적으로The cultivation method of in-flight cultivation of tea tree according to an embodiment of the present invention, specifically
(a) 종피를 제거한 차나무(Camellia sinensis L.) 종자를 멸균하는 단계;(a) sterilizing the seeds of the tea tree ( Camellia sinensis L.) from which the seed skin has been removed;
(b) 상기 (a) 단계에서 멸균한 차나무 종자로부터 유근을 적출하는 단계;(b) extracting the roots from the tea tree seeds sterilized in step (a);
(c) 상기 (b) 단계에서 적출한 유근을 기내(in vitro)에서 배지에 치상하여 신초를 유도하고 1.5~2.5주간 배양하여 차나무 유묘를 생장시키는 단계; 및(c) inducing shoots by placing the roots extracted in step (b) on a medium in vitro and culturing them for 1.5 to 2.5 weeks to grow tea tree seedlings; And
(d) 상기 (c) 단계의 차나무 유묘에 적색광, 청색광 및 백색광이 동일한 광량비율로 혼합되어 이루어진 LED 인공광원을 조사하며 재배하는 단계;를 포함하는 것일 수 있으나, 이에 제한되지 않는다.(d) irradiating and cultivating an LED artificial light source formed by mixing red light, blue light, and white light to the tea tree seedlings of the step (c) at the same light quantity ratio, but is not limited thereto.
상기 (c) 및 (d) 단계의 배양은 24~26℃의 온도 및 14~18 시간/6~10 시간의 명/암 광주기를 가지는 배양기 내에서 배양되는 것일 수 있고, 상기 (d) 단계의 LED 인공광원의 조사 기간은 40~50일 일 수 있으나, 이에 제한되지 않으며, 바람직하게는 청색광 및 백색광이 동일한 광량비율로 혼합되어 이루어진 LED 인공광원을 45일간 조사하며 재배하는 것일 수 있다.The cultivation of steps (c) and (d) may be cultured in an incubator having a temperature of 24 to 26° C. and a light/dark photoperiod of 14 to 18 hours/6 to 10 hours, and of step (d) The irradiation period of the LED artificial light source may be 40 to 50 days, but is not limited thereto, and preferably, an LED artificial light source formed by mixing blue light and white light at the same light quantity ratio may be irradiated for 45 days and cultivated.
본 발명은 또한, 상기 방법에 의해 재배된 뿌리 생육이 증진된 차나무(Camellia sinensis L.) 기내배양묘를 제공한다.The present invention also provides a tea tree ( Camellia sinensis L.) in-flight cultured seedlings with enhanced root growth cultivated by the above method.
본 발명에 따른 재배방법에 의해 재배된 차나무 기내배양묘는 뿌리 수, 뿌리 길이 및 잎의 엽록소 함량 등의 생육 특성이 증진된 것으로, 지하부가 건실하므로 순화 생존율이 우수할 것으로 기대된다. 또한, 기내배양묘는 무균의 무병묘이고, 실생묘와 다르게 형질이 균일한 것이 특징이다.Tea tree in-flight cultured seedlings cultivated by the cultivation method according to the present invention have improved growth characteristics such as the number of roots, root length, and chlorophyll content of leaves, and are expected to have excellent acclimatization survival rate because the underground part is healthy. In addition, in-flight cultured seedlings are sterile and disease-free, and, unlike seedlings, the characteristics are uniform.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by examples. However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited to the following examples.
재료 및 방법Materials and methods
1. 배양재료1. Culture material
본 실험에 사용한 종자는 (재)하동녹차연구소에서 분양받은 하동지역 재래종 차나무 종자를 사용하였다. 차나무 기내 무균 식물체 유도를 위하여 종자의 종피를 제거한 후 70%(v/v) 에탄올에 1분간 침지하여 표면살균하였다. 그 후 Tween 20 (Sigma, USA)을 첨가하여 수돗물로 거품을 내어 씻어낸 다음, 무균상에서 차아염소산나트륨(Sodium hypochlorite, NaClO) (Sigma) 수용액에 3분간 침지시켜 살균하는 과정을 총 3회 실시하고, 멸균증류수로 5회 이상 충분히 세척한 후 필터페이퍼(Advantec, Japan) 위에서 건조시켰다. 그 후 상기 종자에서 유근(radicle)을 적출하여 20㎖의 MS (Murashige & Skoog medium, Duchefa, Netherlands) 고체 배지에 치상하였다. 상기 적출한 유근으로부터 신초를 유도하기 위해 3%(w/v) 자당(Duchefa)과 0.4%(w/v) 겔라이트(Sigma aldrich, USA)가 첨가된 MS 배지를 사용하여 배양하였으며, 상기 MS 배지는 pH 5.6~5.8로 조정한 후 121℃에서 15분간 고압멸균한 후 사용하였다. 배양은 내부온도 25±1℃, 5,000 Lux의 조도로 조절된 형광등을 16/8h (light/dark) 광주기로 조사하며 배양기(Corea science, Korea) 내에서 이루어졌으며, 신초가 유도된 유묘는 상기 배지와 동일 조건의 새로운 배지로 계대배양 하여 2주간 배양 한 다음 모든 실험에 사용하였다.The seeds used in this experiment were from the Hadong-area native tea tree seeds sold in Hadong Green Tea Research Center. In order to induce aseptic plants in the cabin of the tea tree, the seeds were removed and immersed in 70% (v/v) ethanol for 1 minute to sterilize the surface. After that, Tween 20 (Sigma, USA) was added to foam and rinsed with tap water, and then sterilized by immersing in an aqueous solution of sodium hypochlorite (NaClO) (Sigma) for 3 minutes in a sterile phase. , After sufficiently washing 5 or more times with sterile distilled water, it was dried on filter paper (Advantec, Japan). Thereafter, radicles were removed from the seeds and placed on a 20 ml MS (Murashige & Skoog medium, Duchefa, Netherlands) solid medium. In order to induce shoots from the extracted root roots, 3% (w/v) sucrose (Duchefa) and 0.4% (w/v) gellite (Sigma aldrich, USA) were added to incubate using MS medium, and the MS The medium was used after being autoclaved at 121° C. for 15 minutes after adjusting to pH 5.6 to 5.8. Cultivation was carried out in an incubator (Corea science, Korea) by irradiating a fluorescent lamp controlled to an internal temperature of 25±1°C and an illuminance of 5,000 Lux with a 16/8h (light/dark) photoperiod. It was subcultured with a new medium under the same conditions as and cultured for 2 weeks, and then used in all experiments.
2. 광환경2. Light environment
차나무 기내배양 과정 중 증식배양 단계에서의 광질을 조절하여 기내배양묘의 생육에 미치는 영향을 분석하고자 하였다. 전술한 방법을 통해 기내에서 무균의 상태로 유도된 차나무 유묘에 적색광(Red LED, 630nm), 청색광(Blue LED, 460nm) 및 혼합광(Red:Blue:White, 1:1:1)의 LED (W1400×D600×H595, 220V/40W, 고려과학)를 각각 1,000 Lux의 조도로 광조사하였으며, 형광등(Fluorescent Lamp)을 대조구로 하였다. 배양은 내부온도 25±1℃, 광주기 16h/8h (light/dark)의 배양실내에서 이루어 졌으며, 외부광원은 검은 천을 이용하여 차단하였다.The purpose of this study was to analyze the effect on the growth of in-flight seedlings by controlling the light quality at the stage of proliferation during the in-flight culture of tea trees. LEDs of red light (Red LED, 630nm), blue light (Blue LED, 460nm) and mixed light (Red:Blue:White, 1:1:1) on the tea tree seedlings induced in a sterile state in the cabin through the above method ( W1400×D600×H595, 220V/40W, Korea Science) were irradiated with light at an illuminance of 1,000 Lux each, and a fluorescent lamp was used as a control. Cultivation was conducted in a culture chamber at an internal temperature of 25±1℃ and a photoperiod of 16h/8h (light/dark), and the external light source was blocked with a black cloth.
3. 생육인자 측정3. Growth factor measurement
LED 처리에 따른 차나무의 생육특성을 조사하고자 식물의 생육발달 지표가 되는 초장길이(mm), 엽수(No.), 뿌리수(No., 주근+측근 갯수), 뿌리길이(mm, 주근의 길이+측근의 길이), 뿌리단면적(mm2), 뿌리직경(mm), 생채중(mg), 엽록소함량(SPAD value)을 조사하였다. 초장 길이는 베니어캘리퍼스(Absolute IP67, Mitutoyo Co., Japan)를 이용하여 배양배지 지면에서 정단까지의 길이를 측정하였다. 엽록소함량 분석은 엽록소 측정기(SPAD-502 Minolta, Japan)를 이용하여 정단으로부터 2-3번째 엽을 대상으로 측정하였고, 뿌리 형태 분석은 WinRHIZO 프로그램(WinRHIZO Reg 2008a, Regent Instrument Inc., Canada)을 이용하여 조사하였다. 생체중 측정은 모든 실험이 끝난 45일 후 차나무 식물체를 기외로 꺼내 뿌리에 묻은 배지를 완전하게 씻어 낸 후, 식물체의 지상부와 지하부 생체중을 전자저울(EX224G, Ohaus, USA)로 칭량하여 조사하였다. 모든 조사는 5일마다 실시하였으며, 각 처리 당 10본 이상 반복 조사하였다.In order to investigate the growth characteristics of tea trees according to LED treatment, the plant's growth and development indicators are the super-long length (mm), the number of leaves (No.), the number of roots (No., the number of main roots + the number of side roots), the root length (mm, the length of the main roots). + Length of side root), root cross-sectional area (mm 2 ), root diameter (mm), fresh vegetable weight (mg), and chlorophyll content (SPAD value) were investigated. The length of the plant was measured from the surface of the culture medium to the apex using a veneer caliper (Absolute IP67, Mitutoyo Co., Japan). Chlorophyll content analysis was performed using a chlorophyll meter (SPAD-502 Minolta, Japan) for the 2-3rd lobe from the apex, and root morphology analysis was performed using the WinRHIZO program (WinRHIZO Reg 2008a, Regent Instrument Inc., Canada). And investigated. For the measurement of live weight, 45 days after the completion of all experiments, the tea tree plants were taken out of the air and the medium on the roots was completely washed, and the above-ground and underground live weights of the plants were weighed with an electronic scale (EX224G, Ohaus, USA). All investigations were conducted every 5 days, and 10 or more of each treatment were repeated.
4. 광합성 반응 분석4. Photosynthetic reaction analysis
엽 형광이미지는 Portable Handy Cam (FluorCam 1000-H, Photon System Instruments Ltd, Czech Republic)을 이용하여 이미지화하였으며, 모든 식물은 30분간 암적응 후 렌즈와 사이의 거리를 10cm로 동일하게 유지하여 카우츠키유도 방법으로 측정하였다(Kautsky & Hirsch, 1931 Naturwissenschaften. 19(48): 964). 엽록소 형광반응 유도를 위해 낮은 광에서 최초 측정되는 최소 형광값(F0), 그 후 지속적인 포화광을 비추어 최고 형광값(Fm)을 측정하고, 광합성이 진행되는 동안 포화광을 비추어 Fm'을 측정하였다. F0은 광계Ⅱ에 있는 엽록소a 분자가 들뜬 후 에너지가 반응중심으로 이동하기 이전에 나오는 형광을 말하며, 최대 형광값 Fm에서 최소 형광값 F0을 빼준 값을 Fv(=Fm-F0)라고 한다. 또한 엽록소 형광의 비광화학 소멸(nonphotochemical quenching, NPQ)은 아래의 식을 이용하여 산출하였다.The leaf fluorescence image was imaged using a Portable Handy Cam (FluorCam 1000-H, Photon System Instruments Ltd, Czech Republic), and all plants were subjected to dark adaptation for 30 minutes, and the distance between the lens and the lens was kept equal to 10 cm. (Kautsky & Hirsch, 1931 Naturwissenschaften. 19(48): 964). Minimum fluorescence value is first measured at a low light to the chlorophyll fluorescence induction (F 0), then, and in view of the continued saturation optical measuring the maximum fluorescence value (F m), F m 'in view of the saturation light during photosynthesis is in progress Was measured. F 0 refers to the fluorescence emitted after the chlorophyll a molecule in photosystem II is excited and before the energy moves to the reaction center. The value obtained by subtracting the minimum fluorescence value F 0 from the maximum fluorescence value F m is F v (=F m -F It is called 0 ). In addition, nonphotochemical quenching (NPQ) of chlorophyll fluorescence was calculated using the following equation.
NPQ = (Fm - Fm')/Fm'NPQ = (F m -F m ')/F m '
5. 통계분석5. Statistical analysis
실험결과는 SPSS Version 24 (IBA, USA) 프로그램을 사용하여 Duncan's multiple range test 사후검정을 실시하여 평균값 간의 유의차를 비교하였다.For the experimental results, a Duncan's multiple range test post-test was performed using the SPSS Version 24 (IBA, USA) program to compare the significant difference between the mean values.
실시예 1. 생육인자 측정Example 1. Measurement of growth factors
기내배양에서 배지의 성분과 광, 온도 등을 포함하는 물리 화학적인 변화는 식물의 생장뿐 아니라 2차 대사산물의 생합성에 영향을 주기 때문에 배양조건의 확립은 매우 중요하다. 광질에 따른 차나무 기내배양묘 생장양상은 차이를 보였는데, 초장생육은 적색광(Red), 청색광(Blue) 및 혼합광(Red+Blue+White) 처리구에서 대조구(형광등)에 비해 모두 길게 자랐다. 특히, 적색광 처리구(11.77mm)는 대조구(6.21mm)에 비해 줄기 신장이 약 2배 길게 자라는 특징을 보였기 때문에 차나무 기내배양묘의 초장 생육에 있어 효과적인 광질인 것으로 판단되었다.In in-flight culture, the establishment of culture conditions is very important because physicochemical changes including media components, light, temperature, etc. affect not only the growth of plants but also the biosynthesis of secondary metabolites. There was a difference in the growth pattern of in-flight cultured seedlings of tea trees according to the light quality, and the plant growth was all grown longer in the red light (Red), blue light (Blue) and mixed light (Red+Blue+White) treatments than the control (fluorescent lamp). In particular, since the red light treatment group (11.77mm) showed a characteristic that the stem height grows about twice as long as that of the control group (6.21mm), it was judged to be an effective light quality for the plant growth of in-flight tea tree seedlings.
엽수에 있어서는 청색광 처리구(2.93개)에서 대조구(1.96개)에 비해 많이 발생하여 엽생장에 효과적인 것으로 나타났다(표 1). 그러나, 청색광, 혼합광 및 적색광의 모든 처리구에서 엽수는 통계적 유의성이 낮았기 때문에, 처리 기간에 따른 엽생장 비교 등에 대한 추가적인 연구가 필요할 것으로 판단되었다.In leaf trees, it was found to be more effective in leaf growth in the blue light-treated group (2.93 pieces) than in the control group (1.96 pieces) (Table 1). However, in all treatments of blue light, mixed light, and red light, since leaf trees had low statistical significance, it was judged that additional research on leaf growth comparison according to treatment period was necessary.
기내 발근에 있어서 혼합광 처리구는 뿌리 수(2.44개), 길이(9.66mm), 단면적(1.63mm2)이 대조구 및 다른 LED 광원 처리구에 비해 모두 높게 나타나 발근에 효과적인 것을 알 수 있었다(표 1). 반면 청색광 처리구는 뿌리수(1.40개), 길이(4.89mm), 단면적(0.99mm2)에 있어 대조구보다 저조한 것으로 나타났다. 정 등(2009, 한국약용작물학회지. 17(1):39-45)은 가시오갈피(Eleutherococcus senticosus)의 배양에서 적색, 청색 LED 및 형광등 하에서 발근율이 모두 90% 이상으로 높고, 특히 청색광에서는 뿌리가 굵고 솜털같은 세근이 발달되었다고 보고하였다. 또한, 이 등(2014, 한국식물생명공학회지. 2014(41):94-99)은 미선나무(Abeliophyllum distichum) 배양에서 형광등 및 청색광 LED에서 발근이 효과적임을 보고한 바 있다. 상기 보고들을 통해 동일한 광원 예컨대, 청색광이 조사되더라도 식물체의 종류에 따라 생육에 미치는 효과가 상이함을 알 수 있었다.For in-flight rooting, the number of roots (2.44), length (9.66mm), and cross-sectional area (1.63mm 2 ) of the mixed light treatment were higher than that of the control and other LED light source treatments, indicating that it was effective for rooting (Table 1). . On the other hand, the blue light treatment group was found to be inferior to the control in the number of roots (1.40), length (4.89mm), and cross-sectional area (0.99mm 2 ). Jeong, et al. (2009, Journal of the Korean Society of Medicinal Crop Science. 17(1):39-45) reported that the rooting rate of Eleutherococcus senticosus was all higher than 90% under red, blue LED and fluorescent lamps, especially in blue light. It was reported that thick and fluffy fine roots were developed. In addition, Lee et al. (2014, Journal of the Korean Society of Plant Biotechnology. 2014(41):94-99) reported that rooting is effective in fluorescent lamps and blue-light LEDs in culture of Abeliophyllum distichum . Through the above reports, it can be seen that even when the same light source, for example, blue light is irradiated, the effect on growth is different depending on the type of plant.
식물 잎의 엽록소 함량은 묘목의 생리적 상태, 식물체에 흡수된 질소량, 광조건 등에 따라 다양하게 나타난다. 엽록소 함량은 혼합광 처리구에서 가장 높은 값(36.07)이 나타난 반면 대조구에서 가장 낮은값(23.19)이 나타났다(도 1). 즉, 차나무 기내배양 시 혼합광 처리는 엽록소 합성 촉진에 큰 영향을 미치는 것으로 판단되었다. 모든 광질은 식물의 색소형성에 관여하는데, Moreira da Silva 등(1997, Plant Cell, Tissue Organ Cult. 51:187-193)은 청색광이 식물의 엽록소 합성에 중요한 요인이라 보고한 바 있다. 또한 신 등(2008, Acta Physiol. Plant. 30:339-343)은 청색광에서 자란 식물에서 엽록소 함량이 높게 나타난다고 보고한 바 있는데, 본 발명에서는 이전의 보고들과는 다르게 혼합광 처리에서 엽록소 함량이 높아지는 결과를 보였다.The chlorophyll content of plant leaves varies depending on the physiological state of the seedling, the amount of nitrogen absorbed by the plant, and the light conditions. As for the chlorophyll content, the highest value (36.07) appeared in the mixed light treatment group, while the lowest value (23.19) was found in the control group (Fig. 1). That is, it was judged that the mixed light treatment had a great effect on the promotion of chlorophyll synthesis during in-flight culture of tea trees. All minerals are involved in plant pigmentation, and Moreira da Silva et al. (1997, Plant Cell, Tissue Organ Cult. 51:187-193) reported that blue light is an important factor in the synthesis of chlorophyll in plants. In addition, Shin et al. (2008, Acta Physiol. Plant. 30:339-343) reported that the chlorophyll content appeared high in plants grown in blue light, but in the present invention, unlike previous reports, the chlorophyll content increased in mixed light treatment. Showed results.
실시예 2. 광합성 반응 분석Example 2. Photosynthetic reaction analysis
형광반응 이미지는 전체 엽면적을 대상으로 형광 방출을 측정할 수 있으며, 식물의 피해가 엽의 어느 부분에서 진행되며 어떻게 진행되는지를 비파괴적으로 보다 쉽게 알 수 있는 장점이 있다. 엽록소 형광이미지(CFI)는 형광강도에 의한 색의 변화를 근거로 하여 분석하였고, 각 처리 간 형광강도 범위 내에서 파란색은 형광강도가 낮고, 붉은색은 형광강도가 가장 높음을 나타낸다. 도 2의 형광이미지 결과를 통해 혼합광 처리구(도 2D)에서 높은 형광강도인 붉은색이 진하게 띄는 것을 확인할 수 있었고, 청색광 처리구(도 2C)에서 낮은 형광강도인 푸른색이 진하게 띄는 것을 알 수 있었는데, 이러한 결과는 차나무 기내배양 시 혼합광 처리는 청색광, 적색광 및 대조구(형광등)보다 광합성 효율이 높을 수 있음을 시사하였다.Fluorescence reaction images have the advantage of being able to measure fluorescence emission for the entire leaf area, and non-destructively more easily knowing where and how damage to plants proceeds. The chlorophyll fluorescence image (CFI) was analyzed based on the change in color due to the fluorescence intensity, and within the range of fluorescence intensity between treatments, blue indicates that the fluorescence intensity is low, and red indicates that the fluorescence intensity is the highest. Through the fluorescence image result of FIG. 2, it was confirmed that the red color, which is a high fluorescence intensity, stands out in a dark color in the mixed light treatment unit (Fig. 2D), and it can be seen that the blue color, which is a low fluorescence intensity, in the blue light processing unit (Fig. 2C). , These results suggested that the mixed light treatment may have higher photosynthetic efficiency than the blue light, red light and control (fluorescent lamp) during in-flight culture of tea trees.
Fv/Fm은 광계Ⅱ의 활성을 나타내주는 지표이며, 들뜬 에너지 포획능력을 나타낸다. 광질에 따른 차나무 기내배양묘의 Fv/Fm의 값은 혼합광(0.77)>청색광(0.76)>적색광(0.74)>무처리(0.73)의 순으로 나타났으며, 각 처리구간 값의 차이가 거의 없었다. 광계Ⅱ의 활성을 나타내는 Fv/Fm은 비생물학적 스트레스에 대한 중요한 지표로 활용되고 있으며, 일반적으로 건전한 식물의 최대양 자수득률은 0.8 내외로 알려져 있다.F v /F m is an index indicating the activity of photosystem II and indicates the ability to capture excited energy. The values of F v /F m of in-flight cultured seedlings of tea trees according to the light quality were in the order of mixed light (0.77)> blue light (0.76)> red light (0.74)> no treatment (0.73). There was little. F v /F m, which indicates the activity of photosystem II, is used as an important indicator for abiotic stress, and generally, the maximum yield of healthy plants is known to be around 0.8.
한편 비광학적 소실을 의미하는 NPQ의 증가는 일반적으로 내생적, 외생적 스트레스에 대한 반응으로 알려져 있는데, NPQ의 증대는 민감한 수종들에서 나타나며, 내성이 있는 식물에서는 NPQ가 증가하지 않는 것으로 보고되었다. 차나무 기내배양묘의 NPQ 값은 청색광 처리구에서 가장 높은 값(0.92)을 나타냈으며, 무처리 대조구에서 낮은 값(0.30)을 나타냈다(도 3). 상기 결과는 청색광 조건 하에서 엽록소 함량이 낮았던 것에 그 원인이 있는 것으로 보이며, 차나무 기내배양 시 청색광 처리는 배양묘의 광합성 효율 억제를 나타낼 수 있으므로, 적절한 광질의 선택이 양질의 기내배양묘를 재배할 수 있음을 알 수 있었다.Meanwhile, the increase in NPQ, which means non-optical loss, is generally known as a response to endogenous and exogenous stress. The increase in NPQ appears in sensitive species, and it has been reported that NPQ does not increase in resistant plants. The NPQ value of in-flight tea tree seedlings was highest in the blue light treatment group (0.92), and the lowest value (0.30) in the untreated control group (Fig. 3). The above results seem to be due to the low chlorophyll content under blue light conditions, and blue light treatment during in-flight cultivation of tea trees may indicate the suppression of the photosynthetic efficiency of the cultivated seedlings. And it was found.
Claims (5)
(b) 상기 (a) 단계에서 멸균한 차나무 종자로부터 유근을 적출하는 단계;
(c) 상기 (b) 단계에서 적출한 유근을 기내(in vitro)에서 배지에 치상하여 신초를 유도하고 1.5~2.5주간 배양하여 차나무 유묘를 생장시키는 단계; 및
(d) 상기 (c) 단계의 차나무 유묘에 적색광, 청색광 및 백색광이 동일한 광량비율로 혼합되어 이루어진 LED 인공광원을 조사하며 40~50일 동안 재배하는 단계;를 포함하는 것인, 뿌리 수 및 뿌리 길이가 증진된 차나무 기내배양묘의 재배방법.(a) sterilizing the seeds of the tea tree ( Camellia sinensis L.) from which the seed skin has been removed;
(b) extracting the roots from the tea tree seeds sterilized in step (a);
(c) inducing shoots by placing the roots extracted in step (b) on a medium in vitro and culturing them for 1.5 to 2.5 weeks to grow tea tree seedlings; And
(d) irradiating an LED artificial light source formed by mixing red light, blue light, and white light to the tea tree seedlings of step (c) at the same light quantity ratio and cultivating for 40 to 50 days; containing, root number and root Cultivation method of in-flight cultivation of tea tree with increased length.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109618926A (en) * | 2018-11-28 | 2019-04-16 | 西南林业大学 | A method of passing through test tube seedling continuous production tealeaves |
| KR102462676B1 (en) | 2021-10-05 | 2022-11-03 | 권휘 | Method for making tea by using shell of camellia sinensis fruit |
| CN116784237A (en) * | 2023-07-26 | 2023-09-22 | 东北林业大学 | Efficient fraxinus mandshurica regeneration method based on light quality regulation and control |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2015033366A (en) * | 2013-08-09 | 2015-02-19 | シャープ株式会社 | Tea tree raising method and tea tree raising device |
| KR20150076917A (en) * | 2013-12-27 | 2015-07-07 | 전남과학대학교 산학협력단 | Production method of tea-plant |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2015033366A (en) * | 2013-08-09 | 2015-02-19 | シャープ株式会社 | Tea tree raising method and tea tree raising device |
| KR20150076917A (en) * | 2013-12-27 | 2015-07-07 | 전남과학대학교 산학협력단 | Production method of tea-plant |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109618926A (en) * | 2018-11-28 | 2019-04-16 | 西南林业大学 | A method of passing through test tube seedling continuous production tealeaves |
| KR102462676B1 (en) | 2021-10-05 | 2022-11-03 | 권휘 | Method for making tea by using shell of camellia sinensis fruit |
| CN116784237A (en) * | 2023-07-26 | 2023-09-22 | 东北林业大学 | Efficient fraxinus mandshurica regeneration method based on light quality regulation and control |
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