KR101293739B1 - The transformation method of Liriodendron tulipifera embryonic tissue - Google Patents
The transformation method of Liriodendron tulipifera embryonic tissue Download PDFInfo
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Abstract
본 발명은 백합나무 배발생조직의 형질전환 방법에 관한 것으로, 보다 상세하게는 (1) 백합나무(Liriodendron tulipifera)의 배발생(embryogenic) 조직을 아그로박테리움(Agrobacterium)와 공조배양(co-cultivation)하는 제1단계; (2) 상기 제1단계로 부터 수득한 공조배양된 백합나무의 배발생조직에 geneticin 및 cefotaxime을 처리하여 형질전환 배발생조직을 선발하는 제2단계; (3) 상기 제2단계로 부터 수득한 형질전환 배발생조직에 geneticin 및 cefotaxime을 처리하여 체세포배 유도하는 제3단계; 및 (4) 상기 제3단계로 부터 수득한 체세포배에 geneticin 및 cefotaxime을 처리하여 발아식물체를 재분화하는 제4단계; 를 포함하는 백합나무 배발생조직의 형질전환 방법에 관한 것이다.
본 발명은 향후 바이오매스(biomass), 내충성 및 내한성 등의 기능성 백합나무 품종의 창생(創生) 및 그 실용화를 이루기 위한 필수적인 일련의 형질전환 및 그로부터 형질전환체 획득의 기술완성을 위해 필요한 것으로 백합나무 신품종육성 등에 관한 연구에 긍정적인 효과를 부여하는 유리한 효과가 인정된다고 할 것이다.The present invention relates to a method for transforming a pear embryonic tissue, more specifically (1) lily tree ( Liriodendron a first step of a tissue embryogenesis (embryogenic) of tulipifera) Agrobacterium (Agrobacterium) and the air conditioning culture (co-cultivation); (2) a second step of selecting transformed embryogenic tissue by treating geneticin and cefotaxime on the embryogenic tissue of the co-cultured lily tree obtained from the first step; (3) a third step of inducing somatic embryos by treating geneticin and cefotaxime on the transgenic embryonic tissue obtained from the second step; And (4) a fourth step of re-differentiating germinating plants by processing geneticin and cefotaxime on the somatic embryo obtained from the third step; It relates to a method for transforming the embryonic tissue of the lily of the wood containing.
The present invention is necessary for the completion of the technology for the acquisition of transformants and a series of essential transformations for achieving the development and practical use of functional lily tree varieties such as biomass, insect resistance and cold resistance in the future. It will be said that the beneficial effect of giving positive effects to research on new breeding of trees, etc. is recognized.
Description
본 발명은 형질전환에 관한 기술로서 보다 상세하게는 백합나무 배발생조직의 형질기술에 대한 것이다. 한편 본 발명의 발명자는 국책과제인 ‘BT 기반기술을 이용한 백합나무 대량생산 실용화’과제 수행 중 백합나무 배발생조직의 형질전환 기술을 개발하기에 이른 것이다.The present invention is a technique for transformation, and more particularly, it relates to a technique for transforming pear embryonic tissue. On the other hand, the inventor of the present invention has reached the development of a technology for transforming a pear embryonic tissue during the implementation of the national project, “Utilization of mass production of lily trees using BT-based technology”.
백합나무(Liriodendron tulipifera)는 북아메리카가 원산지로 높이가 약 13m에 달하며 나무껍질은 잿빛과 검은빛이 섞인 갈색이다. 잎은 어긋나고 넓고 둥근 달걀 모양이며 길이와 나비는 6∼18cm 정도이다. The lily tree ( Liriodendron tulipifera ) is native to North America, reaching a height of about 13 m and the bark is brown with a mixture of gray and black. The leaves are alternate, broad, rounded egg-shaped, and the length and butterfly are about 6-18cm.
백합나무의 꽃은 5∼6월에 녹색을 띤 노란색으로 피고 가지 끝에 지름 약 6cm의 튤립 같은 꽃이 1개씩 달린다. 꽃받침조각은 3개, 꽃잎은 6개이다. 꽃잎 밑동에는 주황색의 무늬가 있다. The flowers of the lily tree are greenish yellow in May-June, and tulip-like flowers, about 6 cm in diameter, hang at the end of each branch. There are 3 calyx pieces and 6 petals. There is an orange pattern at the base of the petal.
암술과 수술이 많고 꽃이 진 다음 꽃턱이 길이 7cm 정도 자란다. 열매는 폐과로서 10∼11월에 익으며, 날개가 있고 종자가 1∼2개씩 들어 있다. 미국에서는 생장이 빠르므로 중요한 용재수(用材樹)로 쓰나 한국의 중부 이남에서는 관상용으로 심는 것이 일반적이다.
There are many pistils and stamens, and the flower jaws grow to about 7 cm in length. Fruits are Lungaceae, ripening in October-November, with wings and 1 to 2 seeds. Since it grows rapidly in the United States, it is used as an important dragon tree, but it is common to plant it as ornamental in sub-central Korea.
관련 기술로는 본 발명자가 출원하여 등록받은 ‘체세포배 발생을 이용한 백합나무의 번식방법[특허등록번호 : 10-0889342]’ 이 있으며, 이 발명은 체세포배발생을 이용한 백합나무(Liriodendron tulipifera)의 번식방법에 관한 것으로, 보다 상세하게는 백합나무의 번식방법에 있어서, 백합나무 미숙종자배를 배양하여 배발생조직을 유도하고 증식하는 단계, 상기 배발생조직으로부터 체세포배를 유도하는 단계, 상기 체세포배를 발아시켜 식물체로 분화시키키는 단계를 포함하는 체세포배 발생을 이용한 백합나무의 번식방법에 관한 것이다.
As a related technology, there is a method of propagating a lily tree using somatic embryogenesis, which is filed and registered by the present inventor [Patent Registration No.: 10-0889342], and the present invention is made of a lily tree (Liriodendron tulipifera) using somatic embryogenesis. Regarding the propagation method, more specifically, in the method of propagation of a lily tree, the step of inducing and proliferating embryogenic tissue by culturing an immature seedling of a lily tree, inducing somatic embryos from the embryogenic tissue, the somatic cells It relates to a method for propagation of a lily tree using somatic embryogenesis, including the step of germinating the embryo and differentiating it into a plant.
또 다른 관련 기술로는 본 발명자가 출원하여 공개된 ‘백합나무의 체세포배 발생 방법[출원공개번호 : 10-2011-0056056]’이 있으며, 이 발명은 백합나무의 체세포배 발생 방법에 관한 것으로, 보다 상세하게는 백합나무의 종자로부터 배발생조직을 증식하는 단계; 와 상기 증식된 배발생조직을 세포방사 배양법(cell spreading culture)을 이용하여 체세포배 발생시키는 단계;를 포함하는 것을 특징으로 하는 백합나무의 체세포배 발생 방법에 관한 것이다.
Another related technology is the method disclosed by the present inventors and published'Method of generating somatic embryonic cells of a lily of the valley [Application Publication Number: 10-2011-0056056]', and the present invention relates to a method of generating somatic embryogenic cells of a lily tree, More specifically, the step of proliferating embryogenic tissue from the seeds of the lily tree; And generating somatic embryos using the cell spreading culture of the proliferated embryogenic tissues.
상기 관련 기술은 공히 백합나무를 대상으로 하는 점에서는 동일하지만, 본 발명은 배발생 조직의 형질전환을 기술적 특징으로 한다는 점에서 상이하다.Although the above related technologies are the same in that they target lily trees, the present invention is different in that it is characterized by the technical characteristics of the transformation of embryogenic tissues.
본 발명에서는 백합나무 배발생조직의 형질전환 방법을 제공함을 목적으로 한다. 특히 백합나무의 배발생조직의 항생제(geneticin) 내성 임계 농도, 백합나무의 배발생조직과 아그로박테리움과의 형질전환 시간 등 구체적인 조건을 제시함을 목적으로 한다.
It is an object of the present invention to provide a method for transforming pear embryonic tissue. In particular, it aims to present specific conditions such as the critical concentration of antibiotic resistance of the embryogenic tissue of the lily tree, and the transformation time between the embryogenic tissue of the lily tree and Agrobacterium.
본 발명이 이루고자 하는 기술적 과제들은 이상에서 언급한 기술적 과제들로 제한되지 않으며, 언급되지 않은 또 다른 기술적 과제들은 본 발명의 기재로부터 당해 분야에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다. The technical problems to be achieved by the present invention are not limited to the above-mentioned technical problems, and other technical problems not mentioned will be clearly understood by a person having ordinary knowledge in the art from the description of the present invention. .
상기 종래기술의 문제점을 해결하기 위해 본 발명에서는 (1) 백합나무(Liriodendron tulipifera)의 배발생(embryogenic) 조직을 아그로박테리움(Agrobacterium)과 공조배양(co-cultivation)하는 제1단계; (2) 상기 제1단계로 부터 수득한 공조배양된 백합나무의 배발생조직에 geneticin 및 cefotaxime을 처리하여 형질전환 배발생조직을 선발하는 제2단계; (3) 상기 제2단계로 부터 수득한 형질전환 배발생조직에 geneticin 및 cefotaxime을 처리하여 체세포배 유도하는 제3단계; 및 (4) 상기 제3단계로 부터 수득한 체세포배에 geneticin 및 cefotaxime을 처리하여 발아식물체를 재분화하는 제4단계; 를 포함하는 백합나무 배발생조직의 형질전환 방법을 제공한다.
In the present invention to solve the problems of the prior art (1) lily tree ( Liriodendron a first step of embryogenesis (embryogenic) tissue Agrobacterium (Agrobacterium) and the air conditioning culture (co-cultivation) of tulipifera); (2) a second step of selecting transgenic embryogenic tissue by treating geneticin and cefotaxime on the embryogenic tissue of the co-cultured lily tree obtained from the first step; (3) a third step of inducing somatic embryos by treating geneticin and cefotaxime on the transgenic embryonic tissue obtained from the second step; And (4) a fourth step of re-differentiating germinated plants by treating geneticin and cefotaxime on somatic embryos obtained from the third step; It provides a method for transforming the pear embryonic tissue comprising a.
바람직하게는 상기 아그로박테리움은 서열번호 1의 aux promoter, 서열번호 2의 외래 목적 유전자 및 선발표지유전자인 nptII유전자로 구성된 발현벡터 pBI121을 아그로박테리움 LBA 4404에 도입하여 형질전환된 아그로박테리움인 것을 특징으로 할 수 있다.
Preferably, the Agrobacterium is a transformed Agrobacterium by introducing the expression vector pBI121 consisting of the aux promoter of SEQ ID NO: 1, the foreign target gene of SEQ ID NO: 2, and the nptII gene, which is a selection marker gene, into Agrobacterium LBA 4404. It can be characterized by.
바람직하게는 상기 공조배양은 71~73시간인 것을 특징으로 할 수 있다.
Preferably, the air conditioning culture may be characterized in that it is 71 to 73 hours.
보다 상세하게는 형질전환 배발생조직 선발을 위한 상기 제2단계의 geneticin 및 cefotaxime 처리는 (a) 30mg/L 의 geneticin 및 250mg/L cefotaxime 을 2주 동안 처리하는 제a단계; (b) 50mg/L 의 geneticin 및 250mg/L cefotaxime 을 2주 동안 처리하는 제b단계; 및 (c) 70mg/L 의 geneticin 및 250mg/L cefotaxime 을 4~6주 동안 처리하는 제c단계; 를 포함하는 것을 특징으로 할 수 있다.
More specifically, the geneticin and cefotaxime treatment of the second step for the selection of transgenic embryogenic tissue is (a) a step of processing 30mg/L geneticin and 250mg/L cefotaxime for 2 weeks; (b) a step b treatment of 50 mg/L geneticin and 250 mg/L cefotaxime for 2 weeks; And (c) a step c of treating 70 mg/L geneticin and 250 mg/L cefotaxime for 4 to 6 weeks; It may be characterized by including.
보다 상세하게는 체세포배 유도를 위한 상기 제3단계의 geneticin 및 cefotaxime 처리는 30mg/L 의 geneticin 및 250mg/L cefotaxime 을 6~8주 동안 처리하는 것임을 특징으로 할 수 있다.
More specifically, the third step of geneticin and cefotaxime treatment for induction of somatic embryos may be characterized in that 30mg/L geneticin and 250mg/L cefotaxime are treated for 6-8 weeks.
보다 상세하게는 발아식물체 재분화를 위한 상기 제4단계의 geneticin 및 cefotaxime 처리는 50mg/L 의 geneticin 및 250mg/L cefotaxime 을 4주 동안 처리하는 것임을 특징으로 할 수 있다.
More specifically, the fourth step of geneticin and cefotaxime treatment for germination plant re-differentiation may be characterized in that 50mg/L geneticin and 250mg/L cefotaxime are treated for 4 weeks.
한편 본 발명은 상기 방법 중 선택된 어느 하나의 방법으로 형질전환된 백합나무 배발생조직을 제공한다.Meanwhile, the present invention provides a lily-derived embryogenic tissue transformed by any one of the above methods.
본 발명인 백합나무 배발생조직의 형질전환 방법은 백합나무의 배발생조직을 아그로박테리움 (Agrobacterium)과 공조배양 후 형질전환된 조직 유도, 그로부터 체세포배 발생 및 식물체 재분화 (발아체)까지 획득하는 일련의 기술에 관한 것이다.The method of transforming the embryogenic tissue of the lily of the present invention is a series of acquiring the embryonic tissue of the lily tree after incubation with Agrobacterium and inducing the transformed tissue, from which somatic embryogenesis and plant regeneration (germination) It is about technology.
이에 본 발명은 향후 바이오매스(biomass), 내충성 및 내한성 등의 기능성 백합나무 품종의 창생(創生) 및 그 실용화를 이루기 위한 필수적인 일련의 형질전환 및 그로부터 형질전환체 획득의 기술완성을 위해 필요한 것으로 백합나무 신품종육성 등에 관한 연구에 긍정적인 효과를 부여하는 유리한 효과가 인정된다고 할 것이다.Accordingly, the present invention is necessary for the completion of the technology for obtaining a series of transformations and obtaining transformants therefrom in order to achieve the commercialization and practical use of functional lily tree varieties such as biomass, insect resistance and cold resistance in the future. It will be said that the beneficial effect of giving positive effects to research on new breeding of lily trees is recognized.
도 1은 발현벡터 pBI121의 모식도에 관한 것이다.
pAUX : Agrobacterium tumefaciens aux gene promoter
pNOS : Nopaline synthase promoter
NPT II : Neomycin phosphotransferase II
TZS : Trans-zeatin synthase
도 2는 형질전환 예상 배발생조직에 관한 것이다.
도 3은 형질전환된 배발생조직으로부터 체세포배 유도에 관한 것이다.
도 4는 비형질전환 및 형질전환 조직 유래 체세포배 발아 비교에 관한 것이다.
도 5는 외래 DNA PCR 확인한 것이다.(N: non-transgenic, T1～38: Transgenic)
좌 : Primer, T-gene 1/T-gene 2
우 : Primer T-gene 1/TZS 2
도 6은 RT-PCR 활성 비교에 관한 것이다.
도 7은 비형질전환체(왼쪽) 및 형질전환체(오른쪽)의 발아식물체 비교에 관한 것이다.
도 8은 비형질전환체 및 형질전환체의 엽록소 함량 비교에 관한 것이다.
1 : 비형질전환체
2, 3, 4 : 형질전환체1 is a schematic diagram of the expression vector pBI121.
pAUX: Agrobacterium tumefaciens aux gene promoter
pNOS: Nopaline synthase promoter
NPT II: Neomycin phosphotransferase II
TZS: Trans-zeatin synthase
Figure 2 relates to the embryonic tissue expected to be transformed.
3 relates to induction of somatic embryos from transformed embryogenic tissue.
Figure 4 relates to the comparison of somatic embryo germination derived from non-transformed and transformed tissues.
5 is a foreign DNA PCR confirmed. (N: non-transgenic, T1 ~ 38: Transgenic)
Left: Primer, T-
Right: Primer T-
6 relates to a comparison of RT-PCR activity.
Figure 7 relates to the germination of the non-transformants (left) and transformants (right).
Figure 8 relates to the comparison of chlorophyll content of non-transformants and transformants.
1: non-transformant
2, 3, 4: transformants
본 발명은 백합나무 배발생조직의 형질전환 방법에 관한 것으로, 보다 상세하게는 (1) 백합나무(Liriodendron tulipifera)의 배발생(embryogenic) 조직을 아그로박테리움(Agrobacterium)과 공조배양(co-cultivation)하는 제1단계; (2) 상기 제1단계로 부터 수득한 공조배양된 백합나무의 배발생조직에 geneticin 및 cefotaxime을 처리하여 형질전환 배발생조직을 선발하는 제2단계; (3) 상기 제2단계로 부터 수득한 형질전환 배발생조직에 geneticin 및 cefotaxime을 처리하여 체세포배 유도하는 제3단계; 및 (4) 상기 제3단계로 부터 수득한 체세포배에 geneticin 및 cefotaxime을 처리하여 발아식물체를 재분화하는 제4단계; 를 포함하는 백합나무 배발생조직의 형질전환 방법에 관한 것이다.
The present invention relates to a method for transforming the embryogenic tissue of a lily tree, and more specifically, (1) Agrobacterium and co-cultivation of the embryogenic tissue of the lily tree ( Liriodendron tulipifera ). Step 1); (2) a second step of selecting transformed embryogenic tissue by treating geneticin and cefotaxime on the embryogenic tissue of the co-cultured lily tree obtained from the first step; (3) a third step of inducing somatic embryos by treating geneticin and cefotaxime on the transgenic embryonic tissue obtained from the second step; And (4) a fourth step of re-differentiating germinating plants by processing geneticin and cefotaxime on the somatic embryo obtained from the third step; It relates to a method for transforming the embryonic tissue of the lily of the wood containing.
바람직하게는 상기 아그로박테리움은 서열번호 1의 aux promoter, 서열번호 2의 외래 목적 유전자 및 선발표지유전자인 nptII유전자로 구성된 발현벡터 pBI121을 아그로박테리움 LBA 4404에 도입하여 형질전환된 아그로박테리움인 것을 특징으로 할 수 있다.
Preferably, the Agrobacterium is a transformed Agrobacterium by introducing the expression vector pBI121 consisting of the aux promoter of SEQ ID NO: 1, the foreign target gene of SEQ ID NO: 2, and the nptII gene, which is a selection marker gene, into Agrobacterium LBA 4404. It can be characterized by.
바람직하게는 상기 공조배양은 71~73시간인 것을 특징으로 할 수 있다.
Preferably, the air conditioning culture may be characterized in that it is 71 to 73 hours.
보다 상세하게는 형질전환 배발생조직 선발을 위한 상기 제2단계의 geneticin 및 cefotaxime 처리는 (a) 30mg/L 의 geneticin 및 250mg/L cefotaxime 을 2주 동안 처리하는 제a단계; (b) 50mg/L 의 geneticin 및 250mg/L cefotaxime 을 2주 동안 처리하는 제b단계; 및 (c) 70mg/L 의 geneticin 및 250mg/L cefotaxime 을 4~6주 동안 처리하는 제c단계; 를 포함하는 것을 특징으로 할 수 있다.
More specifically, the geneticin and cefotaxime treatment of the second step for the selection of transgenic embryogenic tissue is (a) a step of processing 30mg/L geneticin and 250mg/L cefotaxime for 2 weeks; (b) step b of treating 50 mg/L geneticin and 250 mg/L cefotaxime for 2 weeks; And (c) the c step of treating 70 mg/L geneticin and 250 mg/L cefotaxime for 4 to 6 weeks; It may be characterized by including.
보다 상세하게는 체세포배 유도를 위한 상기 제3단계의 geneticin 및 cefotaxime 처리는 30mg/L 의 geneticin 및 250mg/L cefotaxime 을 6~8주 동안 처리하는 것임을 특징으로 할 수 있다.
More specifically, the third step of geneticin and cefotaxime treatment for induction of somatic embryos may be characterized in that 30mg/L geneticin and 250mg/L cefotaxime are treated for 6-8 weeks.
보다 상세하게는 발아식물체 재분화를 위한 상기 제4단계의 geneticin 및 cefotaxime 처리는 50mg/L 의 geneticin 및 250mg/L cefotaxime 을 4주 동안 처리하는 것임을 특징으로 할 수 있다.
More specifically, the fourth step of geneticin and cefotaxime treatment for germination plant re-differentiation may be characterized in that 50mg/L geneticin and 250mg/L cefotaxime are treated for 4 weeks.
또한 본 발명은 상기 방법 중 선택된 어느 하나의 방법으로 형질전환된 백합나무 배발생조직에 관한 것이다.
In addition, the present invention relates to a lily embryogenic tissue transformed by any one of the methods selected above.
본 발명에 기재된 geneticin은 하기의 화학식(1)과 같은 구조를 갖는 aminoglycoside 항생제(antibiotic)이다.
The geneticin described in the present invention is an aminoglycoside antibiotic having a structure as shown in the following formula (1).
또한 본 발명에 기재된 cefotaxime은 하기의 화학식(2)과 같은 구조를 갖는 cephalosporin 항생제(antibiotic)이다.
In addition, cefotaxime described in the present invention is a cephalosporin antibiotic (antibiotic) having the structure as in the following formula (2).
이하 실험을 통해 검증한 본 발명의 기술적 특징 내지 임계적 의의를 중심으로 본 발명을 구체적으로 설명한다.
Hereinafter, the present invention will be described in detail with reference to technical features or critical significance of the present invention verified through experiments.
[1] 배발생조직의 생장과 geneticin 임계농도와의 관계실험[1] Experimental relationship between embryonic tissue growth and critical concentration of geneticin
상기와 같이 배발생조직의 생장과 geneticin 임계농도와의 관계를 실험한 결과 백합나무 배발생조직의 항생제 (geneticin) 내성 임계농도는 30mg/L 임을 확인할 수 있었다.
As described above, as a result of experimenting the relationship between the growth of embryogenic tissue and the critical concentration of geneticin, it was confirmed that the critical concentration of antibiotic resistance of the embryonic tissue of the lily tree was 30 mg/L.
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[2] Acetosyringon 처리농도에 따른 형질전환 조직 유도 효과 실험[2] Experimental effect of transformed tissue induction according to acetosyringon treatment concentration
상기와 같이 Acetosyringon 처리농도에 따른 형질전환 조직 유도 효과를 실험한 결과 백합나무 배발생조직과 형질전환 시 acetosyringon 처리농도는 0mg/L 임을 알 수 있었다.
As described above, as a result of testing the effect of inducing transformed tissue according to the treatment concentration of Acetosyringon, it was found that the concentration of acetosyringon treatment at the time of transformation with the embryonic tissue of the lily of the wood was 0 mg/L.
[3] Agrobacterium 농도(OD)에 따른 형질전환 조직 유도 효과 실험[3] Experimental effect of inducing transformed tissue according to Agrobacterium concentration (OD)
상기와 같이 Agrobacterium 농도(OD)에 따른 형질전환 조직 유도 효과를 실험한 결과 백합나무 배발생조직과 형질전환 시 Agrobacterium 농도(OD)는 0.5 임을 확인할 수 있었다.
As described above, as a result of experimenting the effect of inducing transformed tissue according to the concentration of Agrobacterium (OD), it was confirmed that the concentration of Agrobacterium (OD) at the time of transformation with the pear embryonic tissue was 0.5.
[4] 공조배양 (co-cultivation) 시간에 따른 형질전환 조직 유도 효과 실험[4] Experimental effect of inducing transformed tissue according to co-cultivation time
상기와 같이 공조배양 시간에 따른 형질전환 조직 유도 효과를 실험한 결과 백합나무 배발생조직과 형질전환 시 Agrobacterium 공조배양(co-cultivation)의 시간은 3일임을 알 수 있었다.As described above, as a result of experimenting the effect of inducing transformed tissue according to the co-cultivation time, it was found that the time of co-cultivation of the Agrobacterium co-cultivation during transformation with the lily-derived embryonic tissue was 3 days.
이상 본 발명의 구체적 실시형태와 관련하여 본 발명을 설명하였으나 이는 예시에 불과하며 본 발명은 이에 제한되지 않는다. 당업자는 본 발명의 범위를 벗어나지 않고 설명된 실시형태를 변경 또는 변형할 수 있으며, 이러한 변경 또는 변형도 본 발명의 범위에 속한다. 또한, 본 명세서에서 설명한 각 구성요소의 물질은 당업자가 공지된 다양한 물질로부터 용이하게 선택하여 대체할 수 있다. 또한 당업자는 본 명세서에서 설명된 구성요소 중 일부를 성능의 열화 없이 생략하거나 성능을 개선하기 위해 구성요소를 추가할 수 있다. 뿐만 아니라, 당업자는 공정 환경이나 장비에 따라 본 명세서에서 설명한 방법 단계의 순서를 변경할 수도 있다. 따라서 본 발명의 범위는 설명된 실시형태가 아니라 특허청구범위 및 그 균등물에 의해 결정되어야 한다.
The present invention has been described above with reference to specific embodiments of the present invention, but this is only an example and the present invention is not limited thereto. Those skilled in the art can change or modify the described embodiments without departing from the scope of the present invention, and such changes or modifications fall within the scope of the present invention. In addition, the material of each component described herein can be easily selected and replaced by a variety of materials known to those skilled in the art. In addition, those skilled in the art may omit some of the components described herein without deterioration of performance or add components to improve performance. In addition, those skilled in the art may change the order of the method steps described herein according to the process environment or equipment. Therefore, the scope of the present invention should be determined not by the described embodiments, but by the claims and equivalents thereof.
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Claims (7)
(2) 상기 제1단계로 부터 수득한 공조배양된 백합나무의 배발생조직에 geneticin 및 cefotaxime을 처리하여 형질전환 배발생조직을 선발하는 제2단계;
(3) 상기 제2단계로 부터 수득한 형질전환 배발생조직에 geneticin 및 cefotaxime을 처리하여 체세포배 유도하는 단계로서, 상기 geneticin 및 cefotaxime 처리는 30mg/L의 geneticin 및 250mg/L cefotaxime을 6~8주 동안 처리하는 제3단계; 및
(4) 상기 제3단계로 부터 수득한 체세포배에 geneticin 및 cefotaxime을 처리하여 발아식물체를 재분화하는 제4단계; 를 포함하는 백합나무 배발생조직의 형질전환 방법.1 lily trees comprising the steps of embryogenesis (embryogenic) tissue Agrobacterium (Agrobacterium) and the air conditioning culture (co-cultivation) of (Liriodendron tulipifera), the Agrobacterium is aux promoter, SEQ ID NO: of SEQ ID NO: 12 Agrobacterium transformed by introducing the expression vector pBI121 composed of the neomycin phosphotransferase II (nptII) gene, which is the foreign target gene and the selection target gene, and having the cleavage map disclosed in FIG. 1 into Agrobacterium LBA 4404 and transformed step;
(2) a second step of selecting transgenic embryogenic tissue by treating geneticin and cefotaxime on the embryogenic tissue of the co-cultured lily tree obtained from the first step;
(3) The step of inducing somatic embryos by treating geneticin and cefotaxime on the transgenic embryonic tissue obtained from the second step, wherein the geneticin and cefotaxime treatments are 30mg/L geneticin and 250mg/L cefotaxime 6-8 A third step of processing during the week; And
(4) a fourth step of re-differentiating germinating plants by treating genetic cells and cefotaxime on somatic embryos obtained from the third step; Method for transforming a pear embryonic tissue comprising a.
The method of claim 1, wherein the air-conditioning culture is 71-73 hours, characterized in that the transforming method of the embryonic tissue of the lily tree.
According to claim 1, wherein the second step of geneticin and cefotaxime treatment for the selection of transformed embryogenic tissue is (a) a step of processing 30mg / L of geneticin and 250mg / L cefotaxime for 2 weeks; (b) step b of treating 50 mg/L geneticin and 250 mg/L cefotaxime for 2 weeks; And (c) the c step of treating 70 mg/L geneticin and 250 mg/L cefotaxime for 4 to 6 weeks; Method of transforming the pear embryonic tissue, characterized in that it comprises a.
Claim 1, claim 3, claim 4 and claim 6, wherein the transformed embryonic tissue transformed by any one of the selected methods.
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