KR100894032B1 - High frequency plant regeneration via somatic embryogenesis in podophyllum peltatum an important medicinal plants for source of anticancer drug - Google Patents

High frequency plant regeneration via somatic embryogenesis in podophyllum peltatum an important medicinal plants for source of anticancer drug Download PDF

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KR100894032B1
KR100894032B1 KR1020070053025A KR20070053025A KR100894032B1 KR 100894032 B1 KR100894032 B1 KR 100894032B1 KR 1020070053025 A KR1020070053025 A KR 1020070053025A KR 20070053025 A KR20070053025 A KR 20070053025A KR 100894032 B1 KR100894032 B1 KR 100894032B1
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Abstract

포도파일로톡신은 3대 항암제 중의 하나로서 주로 폐암, 위암, 대장암, 백혈병과 같은 혈액암에 널리 사용되고 있다. 또한 류마티스 관절염을 위한 새로운 치료제로서도 사용되고 있으며 건선과 같은 피부병뿐만 아니라 말라리아에도 사용되고 있다. 포도파일로톡신은 포도파일럼 헥산드럼 이라는 히말랴야 고산지대 다년생 목본 식물의 지하경 또는 뿌리에서 추출할 수 있었으나 현재 이 식물은 멸종위기식물로 지정되어 있어 거의 찾아보기가 어렵다. 대체 약용식물로서는 북미에서 서식하는 포도파일럼 펠타텀 수종이 각광을 받고 있으나 이 식물 역시 멸종위기 식물로 삽목 같은 방법으로는 번식이 되지 않고 종자 역시 4년에 한번 얻을 수 있지만 종자의 수도 극히 적게 열려 대량번식에 한계가 있다. 따라서 대량번식시킬 수 있는 방법은 식물생명공학기술을 이용한 식물체 기내 번식뿐이다. 본 발명에서는 멸종위기 중요 약용식물인 포도파일럼 펠타텀의 체세포배를 유도한 후 효율적인 식물체 재분화시스템을 확립하여 대량의 포도파일로톡신을 생산할 수 있는 기틀을 마련하고자 하였다. Grape pyrotoxin is one of the three major anticancer agents, and is widely used for blood cancers such as lung cancer, stomach cancer, colon cancer, and leukemia. It is also used as a new treatment for rheumatoid arthritis and is used for malaria as well as skin diseases such as psoriasis. Grape pyrotoxin could be extracted from the roots or roots of perennial woody plants in the Himalayan alpine region called grape pyramid hexane drum, but it is hardly found because it is an endangered plant. As an alternative medicinal plant, a species of grape pyramid Peltatum in North America is in the spotlight, but this plant is also an endangered plant and cannot be reproduced by cutting like a seed and seeds can be obtained once every 4 years, but the number of seeds is very small. There is a limit to mass reproduction. Therefore, the only way to breed a large number of plants is to use the plant biotechnology technology in-flight propagation. In the present invention, after inducing somatic embryos of the endangered medicinal plant, grape pyramid peltatum, to establish an efficient plant regeneration system to provide a framework for producing a large amount of grape pyrotoxin.

포도파일로톡신, 포도파일럼 헥산드럼, 포도파일럼 펠타텀, 체세포배, 재분화시스템 Grape Pyrotoxin, Grape Pyramid Hexane Drum, Grape Pyramid Peltatum, Somatic Embryogenesis, Regeneration System

Description

포도파일로톡신 대량생산을 위한 포도파일럼 펠타텀의 체세포배 유도 및 재분화시스템 확립 {High frequency plant regeneration via somatic embryogenesis in podophyllum peltatum an important medicinal plants for source of anticancer drug}High frequency plant regeneration via somatic embryogenesis in podophyllum peltatum an important medicinal plants for source of anticancer drug

도 1 은 실시예 1 내지 5에 의하여 포도파일럼 식물조직으로부터 체세포배 발생 캘러스 유도 및 식물체 전환의 결과를 나타낸 사진이다.1 is a photograph showing the results of somatic embryo development callus induction and plant conversion from the grape pile plant tissues according to Examples 1 to 5.

임상에서 사용되고 있는 3대 항암제 중의 하나인 포도파일로톡신의 대량생산을 위한 포도파일럼 헥산드럼의 연구는 오래전부터 진행되었다. 포도파일럼 헥산드럼의 종자에서 체세포배유도(Arumugam et al.1989), 바이오리엑터를 이용한 포도파일럼 헥산드럼의 세포배양(Chattopadhyay et al.2002), 포도파일로톡신의 생산증가를 위한 현탁배양조건 (Chattopadhyay et al.2003)실험 등이 이루어졌지만 성공적인 실용화는 이루어지지 않았고 주로 세포배양에 관한 실험만이 이루어졌다.포도파일로톡신의 경우는 포도파일럼 헥산드럼의 뿌리에서 많이 추출되기 때문에 부정근이나 모상근의 유도가 적절하다고 생각된다.The study of grape pyramid hexane drums for mass production of grape pyrotoxin, one of the three anticancer drugs used in the clinic, has been conducted for a long time. Somatic cell induction from seed of grape pile hexane drum (Arumugam et al. 1989), cell culture of grape pile hexane drum using bioreactor (Chattopadhyay et al. 2002), suspension culture for increased production of grape pyrotoxin Conditions (Chattopadhyay et al. 2003) have been tested, but no practical application has been made, and only cell culture experiments have been carried out. Induction of hairy roots is appropriate.

하지만 지금까지는 부정근이나 모상근에 대한 연구가 미약하였고 포도파일럼 펠타텀의 경우 위와 같은 연구가 전무 하며 멸종위기 식물로 지정되어 있어 식물생명공학적 접근 방법이 매우 시급한 상황이다. 포도파일럼 펠타텀의 잎에서의 포도파일로톡신 함량이 높기 때문에 체세포 배를 유도한 후 식물체로 재분화시키는 연구는 필요하다고 생각된다.However, until now, research on inferior or hairy roots has been poorly conducted, and in the case of grape pyramid peltatum, none of the above studies have been designated as an endangered plant, so the plant biotechnological approach is very urgent. Because of the high content of grape pyrotoxin in the leaves of grape pylum feltatum, it is considered necessary to induce somatic embryos and re-differentiate them into plants.

본 연구는 포도파일럼 펠타텀의 대량생산을 위한 효율적인 식물체 재분화시스템 확립을 위한 것으로 고빈도 고 효율적인 식물체 재분화시스템 확립을 위해 최적의 배양환경을 구축하고 나아가 이러한 기술력을 바탕으로 부정근 모상근 유도 및 포도파일로톡신의 고 함유 식물체 개발을 목적으로 하고 있다. The purpose of this study is to establish an efficient plant regeneration system for mass production of grape pyramid feltatum. It aims at the development of high content of rotoxins.

본 발명의 포도파일로톡신 대량 생산을 위한 포도파일럼 펠타텀의 재분화시스템 확립방법은, 1) 포도파일럼 펠타텀(Podophyllum peltatum) 종자의 접합자배 또는 접합자배의 자엽을 MS(Murashige and Skoog 1962) 배지에 30g/l 수크로오스, 0.8% 젤라이트, NAA 21.6 μM가 첨가된 배지에 치상하여 체세포배를 유도하는 단계, 2) 상기 1) 단계에서 유도된 체세포배를 MS 배지에 30g/l 수크로오스, 0.8% 젤라이트 및 6.78 ~ 9.04μM 2,4-D가 첨가된 배지에 치상하여 체세포배 발생 캘러스를 유도하는 단계. 3) 상기 2) 단계에서 유도된 체세포배 발생 캘러스를 MS 배지에 30g/l 수크로오스, 0.8% 젤라이트 및 3.78 ~ 18.9μM ABA가 첨가된 배지에 치상하여 체세포배를 얻는 단계, 및, 4) 상기 3) 단계에서 얻어진 체세포배 중 자엽시기의 체세포배를 MS배지에 2.89μM GA3 를 첨가한 배지에 치상 및 발아시켜 식물체를 생산하는 단계를 포함하여 이루어지는 것을 특징으로 한다.
이하 본 발명의 방법을 구체적으로 설명하면 다음과 같다.The method for establishing a regeneration system of grape pyramid feltatum for mass production of grape pyrotoxin of the present invention comprises: 1) MS (Murashige and Skoog 1962) of the cotyledon or cotyledon of the seedling of Pyramidal Peltatum A) inducing somatic embryos on the medium to which 30 g / l sucrose, 0.8% zeolite, and 21.6 μM of NAA are added to the medium, 2) 30 g / l sucrose to MS medium, Inducing somatic embryogenic callus by densification in medium to which 0.8% gelite and 6.78 to 9.04 μM 2,4-D were added. 3) obtaining somatic embryos by soaking the somatic embryo generating callus induced in step 2) in a medium to which 30 g / l sucrose, 0.8% zeolite and 3.78-18.9 μM ABA are added to MS medium, and 4) the Among the somatic embryos obtained in step 3), the somatic embryo of the cotyledon phase is densified and germinated in a medium to which 2.89 μM GA 3 is added to the MS medium to produce a plant.
Hereinafter, the method of the present invention will be described in detail.

[실시예 1] 접합자배의 자엽으로부터 체세포배를 유도한 후 유도된 체세포배의 자엽에서 다시 체세포발생 캘러스를 유도. Example 1 Somatic embryos were induced from the cotyledon of the zygote embryo, and somatic embryogenic callus was induced again from the cotyledon of the induced somatic embryo.

포도파일럼 펠타텀의 종자를 4℃에서 한 달 동안 저온 처리한 후 70% Et-OH에서 1분간, 소디움 하이포크로라이드에서 20분간 멸균한 후 멸균수로 5번 세척한 후 종자의 배 부분만 잘라 MS(Murashige and Skoog 1962)배지에, 30g/L의 수크로오스와 4g/L의 젤라이트를 첨가하여 조성된 배지에서 2주 발아시켰다. 발아된 종자로부터 접합자배를 무균적으로 꺼내어 접합자배를 그대로 배양하거나 자엽만을 절취하여 MS 배지에, 30g/L의 수크로오스와 8g/L의 젤라이트를 첨가하여 조성되며, 여기에 NAA의 함량이 각각 0μM, 5.4μM, 10.8μM, 21.6μM, 및 43.2μM으로 첨가된 각각의 배지에 치상한 후 8주 후 체세포배발생률을 확인하였다. 이렇게 유도된 체세포배의 자엽 절편을 다시 MS 배지에, 30g/L의 수크로오스와, 8g/L의 젤라이트를 첨가하여 조성되며, 여기에 2,4-D의 함량이 각각 0.0μM, 2.26μM, 4.52μM, 6.78μM, 9.04μM, 및 11.30μM으로 첨가된 각각의 배지에 치상하여 체세포배 발생 캘러스를 유도하였다.
모든 식물은 한 개체의 식물일지라도 식물의 부위에 따라서 생리적 연령이 다르며 식물체의 노화에 따라 체세포발생 캘러스 유도가 매우 어려울 경우가 있다. 따라서 빠르고 효율적인 체세포 발생 캘러스 유도를 위해 본 연구는 종자의 자엽에서 체세포배를 유도한 후 그 체세포 배의 자엽을 이용, 고효율의 체세포배 발생 캘러스를 유도하는데 성공 하였다.(표 1, 2) After cooling the seed of grape pyramid Peltatum for one month at 4 ° C, sterilize it for 1 minute in 70% Et-OH and 20 minutes in sodium hypochloride, and then wash it 5 times with sterile water. The germinated MS (Murashige and Skoog 1962) medium was germinated for 2 weeks in a medium prepared by adding 30 g / L sucrose and 4 g / L gelite. The germ pears are germinated aseptically from germinated seeds and cultured as is, or the cotyledons are cut out and added to the MS medium by adding 30 g / L sucrose and 8 g / L gelite, each containing NAA content. The somatic embryogenesis rate was confirmed 8 weeks after being wound on each medium added at 0 μM, 5.4 μM, 10.8 μM, 21.6 μM, and 43.2 μM. The cotyledon slices of the somatic embryos thus obtained were formed by adding 30 g / L of sucrose and 8 g / L of zeolite to MS medium, where the contents of 2,4-D were 0.0 μM, 2.26 μM, Somatic embryogenic callus was induced by immunization with each medium added at 4.52 μM, 6.78 μM, 9.04 μM, and 11.30 μM.
All plants have different physiological ages depending on the parts of the plant, even if they are plants of one individual, and it is very difficult to induce somatic cell callus according to the aging of the plant. Therefore, in order to induce somatic embryonic callus for fast and efficient, this study succeeded in inducing high efficiency somatic embryogenic callus by inducing somatic embryos from seed cotyledon.

표1. Table 1.

포도파일럼 펠타텀 접합자배 자엽으로부터 체세포배발생을 위한 최적의 NAA농도 (MS 배지에 30g/L의 수크로오스와 8g/L의 젤라이트를 첨가함)Optimum NAA Concentration for Somatic Embryogenesis from Cotyledon Filamentous Cotyledon Cotyledon (Added 30g / L Sucrose and 8g / L Gelite to MS Medium)

배지조성  Badge composition 접합자 자엽 한개당 발생된 체세포배 수 Number of somatic embryos generated per cotyledon MS(NAA 0μM)  MS (NAA 0 μM) 0 0 MS(NAA 5.4μM)  MS (NAA 5.4 μM) 0 0 MS(NAA 10.8μM)  MS (NAA 10.8 μM) 4 4 MS(NAA 21.6μM)  MS (NAA 21.6 μM) 12 12 MS(NAA 43.2μM)  MS (NAA 43.2 μM) 0 0

표2. 유도된 체세포배의 자엽으로부터 체세포발생 캘러스 발생률 증가를 위한 최적 2,4-D농도 (MS 배지에 30g/L의 수크로오스와 8g/L의 젤라이트를 첨가함) Table 2. Optimal 2,4-D concentration for increasing the incidence of somatic cell callus from cotyledons of induced somatic embryos (30 g / L sucrose and 8 g / L gelite added to MS medium)

배지조성  Badge composition 체세포배의 자엽으로부터 체세포캘러스발생률(%)  Incidence of somatic callus from cotyledon of somatic embryo (%) MS(2,4-D 0μM)  MS (2,4-D 0 μM) 0 0 MS(2,4-D 2.26μM)  MS (2,4-D 2.26 μM) 21 21 MS(2,4-D 4.52μM)  MS (2,4-D 4.52 μM) 45 45 MS(2,4-D 6.78μM)  MS (2,4-D 6.78 μM) 72  72 MS(2,4-D 9.04μM)  MS (2,4-D 9.04 μM) 82 82

[실시예 2]체세포 발생 캘러스로부터 체세포배 유도Example 2 Induction of Somatic Embryos from Somatic Cell Callus

유도된 체세포배 발생 캘러스를 MS 배지에 30g/L의 수크로오스와 8g/L의 젤라이트를 포함하여 조성되는 배지에, ABA가 각각 0.00μM, 3.78μM, 11.35μM, 18.9μM, 및 37.8μM 첨가된 배지에 각각 치상한 후 체세포 배를 유도하였다. 결과 ABA 11.35μM처리구에서 한 캘러스 덩어리당 15개의 체세포배를 얻는 데 성공하였다.Induced somatic embryogenic callus was added to the medium containing 30 g / L sucrose and 8 g / L zeolite in MS medium, with ABA added at 0.00 μM, 3.78 μM, 11.35 μM, 18.9 μM, and 37.8 μM, respectively. After soaking in the medium, somatic embryos were induced. Results We obtained 15 somatic embryos per callus mass in ABA 11.35 μM treatment.

표 3.정상적인 체세포배 유도를 위한 ABA 최적 농도Table 3.Optimal ABA Concentrations for Normal Somatic Embryogenesis

체세포캘러스에서 유도된 체세포배 수  Somatic Embryogenesis Derived from Somatic Callus 배지조건  Badge condition 정상 체세포배수 Normal Somatic Drainage 비정상 체세포배수 Abnormal Somatic Drainage 총체세포배수  Total Cell Drainage MS(ABA 0μM)  MS (ABA 0 μM) 9.3±0.6 9.3 ± 0.6 4.4±0.3 4.4 ± 0.3 13.7 13.7 MS(ABA 3.78μM)  MS (ABA 3.78 μM) 13.6±0.8 13.6 ± 0.8 1.3±0.3 1.3 ± 0.3 14.9 14.9 MS(ABA 11.35μM)  MS (ABA 11.35 μM) 15.3±0.8 15.3 ± 0.8 0 0 15.3 15.3 MS(ABA 18.9μM)  MS (ABA 18.9 μM) 8.3±0.8 8.3 ± 0.8 0 0 8.3 8.3 MS(ABA 37.8μM)  MS (ABA 37.8 μM) 0 0 0 0 0 0

[실시예 3]체세포배로부터 식물체로 변환Example 3 Conversion from Somatic Embryos to Plants

유도된 자엽시기의 체세포배를 MS배지에 ABA 11.35μM와 GA3 2.89μM가 각각 첨가된 배지와, 호르몬을 전혀 처리하지 않은 배지에 각각 치상한 후 배의 발아도(성숙도)를 측정하였다. 결과 GA3 2.89μM첨가 처리구에서 건실하게 발아된 체세포배가 76% 유도되었다.Somatic embryos of the induced cotyledons were implanted into medium containing ABA 11.35 μM and GA 3 2.89 μM, respectively, and medium without hormones, and embryo germination (maturation) was measured. Results 76% of well germinated somatic embryos were induced in the GA 3 2.89 μM addition treatment.

표 4.유도된 체세포의 발아율 증대를 위한 배지조건 Table 4. Medium condition for increasing germination rate of induced somatic cells

배지조건  Badge condition 체세포배의 발아도(%) Germination degree of somatic embryo (%) MS(호르몬 무첨가)  MS (No Hormone) 46±2.5 46 ± 2.5 MS(GA3 2.89μM첨가) MS (GA 3 2.89μM addition) 76±2.1 76 ± 2.1 MS(ABA 11.35μM첨가)  MS (ABA 11.35μM addition) 0 0

[실시예 4]성공적 토양순화를 위한 뿌리생장 조건확립Example 4 Establishment of Root Growth Conditions for Successful Soil Purification

지베렐린(Gibberellric acid) 2.89μM가 첨가된 배지에서 성숙된 자엽시기의 배를 MS 배지를 1/2희석 한 후 15g/L의 수크로오스를 첨가한 배지에 치상하여 왕성한 뿌리를 유도하였는데 성공하였다. In the medium supplemented with Gibberellric acid (2.89 μM), mature cotyledon pears were diluted with 1/2 medium of MS medium, and then sprinkled on medium containing 15 g / L sucrose to induce vigorous roots.

[실시예 5]토양순화 조건확립Example 5 Soil Purification Conditions Established

토양 순화를 극대화하기 위해 멸균된 펄라이트와 피트모스, 버미큘라이트(1:1:1 v/v/v)의 pH를 5.7로 맞춘 후 뿌리가 잘 자란 포도파일럼 펠타텀 유식물체를 이식 하였다.In order to maximize soil purification, the pH of sterile pearlite, peat moss, and vermiculite (1: 1: 1 v / v / v) was adjusted to 5.7, and the well-grown grape pyramid Peltatum seedlings were transplanted.

포도파일럼 펠타텀과 같은 식물의 경우, 항암제의 중요 원료가 되면서 멸종위기로  In the case of plants such as grape pile peltatum, it becomes an important raw material of anticancer drugs and endangered

인해 공급이 절대적으로 부족한 경우 생명공학기술을 이용한 기내번식기술은 유일Due to the absolute shortage of supplies, the only in-flight breeding technology using biotechnology

한 대안이라고 할 수 있다. 이미 임상에서 사용되고 있어 포도파일로톡신의 효과가It is an alternative. It is already used in the clinic, so the effect of grape pyrotoxin

입증되었기 때문에 본 연구의 성공 효과는 세계적인 항암제 시장에 커다란 영향을 The proven success of this study has had a profound impact on the global anticancer market.

미칠 수 있다고 여겨진다. It seems to be crazy.

Claims (3)

삭제delete 삭제delete 1) 포도파일럼 펠타텀(Podophyllum peltatum) 종자의 접합자배 또는 접합자배의 자엽을 MS(Murashige and Skoog 1962) 배지에 30g/l 수크로오스, 0.8% 젤라이트, NAA 21.6 μM가 첨가된 배지에 치상하여 체세포배를 유도하는 단계,1) Conjugate or cotyledons of the seedlings of Podophyllum peltatum seeds were densified on medium supplemented with 30 g / l sucrose, 0.8% gelite and NAA 21.6 μM in MS (Murashige and Skoog 1962) medium. Inducing somatic embryos, 2) 상기 1) 단계에서 유도된 체세포배를 MS 배지에 30g/l 수크로오스, 0.8% 젤라이트 및 6.78 ~ 9.04μM 2,4-D가 첨가된 배지에 치상하여 체세포배 발생 캘러스를 유도하는 단계.2) inducing somatic embryogenic callus by soaking the somatic embryo induced in step 1) in a medium to which 30 g / l sucrose, 0.8% zeolite and 6.78 to 9.04 μM 2,4-D are added to MS medium. 3) 상기 2) 단계에서 유도된 체세포배 발생 캘러스를 MS 배지에 30g/l 수크로오스, 0.8% 젤라이트 및 3.78 ~ 18.9μM ABA가 첨가된 배지에 치상하여 체세포배를 얻는 단계, 및,3) obtaining somatic embryos by soaking the somatic embryo generating callus induced in step 2) in a medium to which 30 g / l sucrose, 0.8% zeolite and 3.78 to 18.9 μM ABA are added to MS medium, and 4) 상기 3) 단계에서 얻어진 체세포배 중 자엽시기의 체세포배를 MS 배지에 2.89μM GA3 를 첨가한 배지에 치상 및 발아시켜 식물체를 생산하는 단계4) producing plants by denting and germinating somatic embryos of the cotyledon season among the somatic embryos obtained in step 3) in a medium to which 2.89 μM GA 3 is added to MS medium 를 포함하여 이루어지는 것을 특징으로 하는 포도파일로톡신 대량 생산을 위한 포도파일럼 펠타텀의 재분화시스템 확립방법.Method for establishing a regeneration system of grape pyramid feltatum for mass production of grape pyrotoxin, characterized in that comprises a.
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