KR20130014942A - 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
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 relates to the technology of transformation, and more specifically, to the transformation technology of lily tree embryogenic tissue. On the other hand, the inventor of the present invention is to develop the transformation technology of the lily embryonic embryonic tissue during 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. Liriodendron tulipifera is native to North America and is about 13m high and its bark is brown with ash and black color. Leaves are alternate, wide, round egg-shaped, about 6-18cm in length and butterfly.
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. Lily flowers bloom greenish yellow in May-June, with one tulip-like flower about 6cm in diameter. 3 sepals and 6 petals. The base of the petal has an orange pattern.
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 after flowering, the jaws grow about 7cm long. Fruits are lungs, ripen in October-November, with wings and 1-2 seeds. It grows fast in the United States, so it is used as an important spring tree, but it is common to plant it for ornamental use 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 art, the present inventors have applied for and registered a 'method of breeding a lily tree using somatic embryogenesis [patent registration number: 10-0889342]', and the present invention provides a method for producing a lily tree using somatic embryogenesis (Liriodendron tulipifera). Regarding the propagation method, and more particularly in the method of propagating the lily tree, the step of inducing and propagating embryogenic tissue by culturing a lily immature seed embryo, inducing somatic embryos from the embryogenic tissue, the somatic cells It relates to a method of propagating a lily tree using a somatic embryo development comprising the step of germinating embryos to differentiate them into plants.
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.
In another related art, there is a method for generating somatic embryos of lily tree, which is disclosed and applied by the present inventors. The present invention relates to a method for generating somatic embryos of a lily tree. More specifically, the step of propagating embryogenic tissue from the seed of the lily tree; And somatic embryogenesis using the cell spreading culture of the propagated embryonic tissue.
상기 관련 기술은 공히 백합나무를 대상으로 하는 점에서는 동일하지만, 본 발명은 배발생 조직의 형질전환을 기술적 특징으로 한다는 점에서 상이하다.Although the related art is the same in that it targets the yellow-poplar tree, the present invention is different in that the transformation of embryogenic tissues is a technical feature.
본 발명에서는 백합나무 배발생조직의 형질전환 방법을 제공함을 목적으로 한다. 특히 합나무의 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 a lily tree embryogenic tissue. In particular, it aims to present specific conditions such as the threshold of antibiotic resistance (geneticin) resistance of the embryonic tissues of the synapse and the time of transformation between the embryonic tissues of Asteraceae 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 objects to be achieved by the present invention are not limited to the above-mentioned technical problems, and other technical subjects which are not mentioned can be clearly understood by those skilled 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) Lilies ( 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 a transgenic embryonic tissue by treating genetic embryo and cefotaxime to the embryogenic tissue of the co-cultured lily tree obtained from the first step; (3) a third step of inducing somatic embryos by treating genetic embryos and cefotaxime to the transgenic embryonic tissue obtained from the second step; And (4) a fourth step of regenerating the germinated plant by treating genetic somatic embryos obtained from the third step with geneticin and cefotaxime; It provides a method for transforming a lily tree embryogenic 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 transformed into an Agrobacterium LBA 4404 by introducing an 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, a selection marker gene, to Agrobacterium LBA 4404. It may be characterized by.
바람직하게는 상기 공조배양은 71~73시간인 것을 특징으로 할 수 있다.
Preferably the co-culture may be characterized in that 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 second step of geneticin and cefotaxime treatment for selection of transgenic embryonic tissues comprises: (a) a step of treating 30 mg / L of geneticin and 250 mg / L cefotaxime for 2 weeks; (b) step b of 50 mg / L geneticin and 250 mg / L cefotaxime for 2 weeks; And (c) treating step 70c / L with geneticin and 250mg / L cefotaxime for 4-6 weeks; It may be characterized in that it comprises a.
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.
In more detail, the third step of geneticin and cefotaxime treatment for inducing somatic embryos may be characterized by treatment of 30 mg / L geneticin and 250 mg / L cefotaxime 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 regeneration may be characterized by treatment of 50 mg / L of geneticin and 250 mg / L cefotaxime for 4 weeks.
한편 본 발명은 상기 방법 중 선택된 어느 하나의 방법으로 형질전환된 백합나무 배발생조직을 제공한다.Meanwhile, the present invention provides a lily tree 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.In the present invention, a method of transforming a lily tree embryogenic tissue is obtained by inducing a transformed tissue after coculture with Agrobacterium ( Agrobacterium ), from which somatic embryo development and plant regeneration (germ) are obtained. It's 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.Therefore, the present invention is necessary for the completion of a series of transformations necessary for achieving the emergence and functionalization of functional lily tree varieties such as biomass, insect resistance and cold resistance, and to complete the technology for obtaining transformants therefrom. The beneficial effect of giving positive effects to the study on new breeding of the lily tree will be recognized.
도 1은 pAUX 유전자벡터 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 및 형질전환체의 기관 분화율 비교에 관한 것이다.
1 : 비형질전환체
2, 3, 4 : 형질전환체
도 8은 비형질전환체(왼쪽) 및 형질전환체(오른쪽)의 발아식물체 비교에 관한 것이다.
도 9는 비형질전환체 및 형질전환체의 기관 길이 비교에 관한 것이다.
1 : 비형질전환체
2, 3, 4 : 형질전환체
도 10은 비형질전환체 및 형질전환체의 엽록소 함량 비교에 관한 것이다.
1 : 비형질전환체
2, 3, 4 : 형질전환체1 is a schematic diagram of the pAUX gene vector.
pAUX: Agrobacterium tumefaciens aux gene promoter
pNOS: Nopaline synthase promoter
NPT II: Neomycin phosphotransferase II
TZS: Trans - zeatin synthase
Figure 2 relates to the expected transformed embryonic tissue.
3 relates to the induction of somatic embryos from transformed embryonic tissues.
4 relates to a comparison of non-transformed and transformed tissue-derived somatic embryo germination.
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.
7 relates to comparison of organ differentiation rate of non-transformers and transformants.
1: non-transformer
2, 3, 4: transformants
FIG. 8 relates to the germination of nontransformants (left) and transformants (right).
9 relates to organ length comparison of non-transformers and transformants.
1: non-transformer
2, 3, 4: transformants
10 relates to a comparison of chlorophyll content of non-transformers and transformants.
1: non-transformer
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 a lily embryogenic tissue, and more specifically, (1) co-cultivation of Agrobacterium ( Agrobacterium ) with the embryogenic tissue of Liriodendron tulipifera A first step); (2) a second step of selecting a transgenic embryonic tissue by treating genetic embryo and cefotaxime to the embryogenic tissue of the co-cultured lily tree obtained from the first step; (3) a third step of inducing somatic embryos by treating genetic embryos and cefotaxime to the transgenic embryonic tissue obtained from the second step; And (4) a fourth step of regenerating the germinated plant by treating genetic somatic embryos obtained from the third step with geneticin and cefotaxime; It relates to a method for transforming a lily 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 transformed into an Agrobacterium LBA 4404 by introducing an 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, a selection marker gene, to Agrobacterium LBA 4404. It may be characterized by.
바람직하게는 상기 공조배양은 71~73시간인 것을 특징으로 할 수 있다.
Preferably the co-culture may be characterized in that 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 second step of geneticin and cefotaxime treatment for selection of transgenic embryonic tissues comprises: (a) a step of treating 30 mg / L of geneticin and 250 mg / L cefotaxime for 2 weeks; (b) step b of 50 mg / L geneticin and 250 mg / L cefotaxime for 2 weeks; And (c) treating step 70c / L with geneticin and 250mg / L cefotaxime for 4-6 weeks; It may be characterized in that it comprises a.
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.
In more detail, the third step of geneticin and cefotaxime treatment for inducing somatic embryos may be characterized by treatment of 30 mg / L geneticin and 250 mg / L cefotaxime 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 regeneration may be characterized by treatment of 50 mg / L of geneticin and 250 mg / L cefotaxime for 4 weeks.
또한 본 발명은 상기 방법 중 선택된 어느 하나의 방법으로 형질전환된 백합나무 배발생조직에 관한 것이다.
The present invention also relates to a lily tree embryogenic tissue transformed by any one of the above methods.
본 발명에 기재된 geneticin은 하기의 화학식(1)과 같은 구조를 같는 aminoglycoside 항생제(antibiotic)이다.
The geneticin described in the present invention is an aminoglycoside antibiotic (bibiotic) having the same structure as in formula (1) below.
또한 본 발명에 기재된 cefotaxime은 하기의 화학식(2)과 같은 구조를 같는 cephalosporin 항생제(antibiotic)이다.
In addition, cefotaxime described in the present invention is a cephalosporin antibiotic (bibiotic) having the same structure as the 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 based on the technical features and critical significance of the present invention verified through experiments.
[1] 배발생조직의 생장과 geneticin 임계농도와의 관계실험[1] Relationship between growth of embryogenic tissue and geneticin critical concentration
상기와 같이 배발생조직의 생장과 geneticin 임계농도와의 관계를 실험한 결과 백합나무 배발생조직의 항생제 (geneticin) 내성 임계농도는 30mg/L 임을 확인할 수 있었다.
As a result of the relationship between the growth of the embryogenic tissue and the critical concentration of geneticin, it was confirmed that the threshold of antibiotic resistance (geneticin) resistance of the lily embryonic tissue was 30 mg / L.
[2] Agrobacterium과의 형질전환 시간에 따른 형질전환 조직 유도 효과 실험[2] Transgenic Tissue Induction Effect of Transgenic Time with Agrobacterium
상기와 같이 Agrobacterium과의 형질전환 시간에 따른 형질전환 조직 유도 효과를 실험한 결과 백합나무 배발생조직과 Agrobacterium과의 형질전환 시간은 30분임을 확인할 수 있었다.
As a result of testing the transformed tissue induction effect according to the transformation time with Agrobacterium as described above, it was confirmed that the transformation time between the lily germ cells and Agrobacterium was 30 minutes.
[3] Acetosyringon 처리농도에 따른 형질전환 조직 유도 효과 실험[3] Transformation Tissue Induction Effect of Acetosyringon 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 a result of testing the transformed tissue induction effect according to Acetosyringon treatment concentration, it was found that the acetosyringon treatment concentration at the time of transformation and the lily embryogenic tissue is 0mg / L.
[4] Agrobacterium 농도(OD)에 따른 형질전환 조직 유도 효과 실험[4] Transgenic Tissue Induction Effect 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 a result of testing the transformed tissue induction effect according to the Agrobacterium concentration (OD) it was confirmed that the Agrobacterium concentration (OD) is 0.5 when transformed with the lily embryogenic tissue.
[5] 공조배양 (co-cultivation) 시간에 따른 형질전환 조직 유도 효과 실험[5] Transgenic Tissue Induction Effect with 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 a result of testing the transformed tissue induction effect according to the co-culture time as described above, it was found that the time of co-cultivation of Agrobacterium co-cultivation during the transformation with the lily embryogenic tissue.
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.
Although the present invention has been described in connection with the specific embodiments of the present invention, it is to be understood that the present invention is not limited thereto. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. In addition, the materials of each component described herein can be readily selected and substituted for various materials known to those skilled in the art. Those skilled in the art will also appreciate that some of the components described herein can be omitted without degrading performance or adding components to improve performance. In addition, those skilled in the art may change the order of the method steps described herein depending on the process environment or equipment. Therefore, the scope of the present invention should be determined by the appended claims and equivalents thereof, not by the embodiments described.
<110> Korea Forest Research Institute(KFRI) <120> The transformation method of Liriodendron tulipifera embryonic tissue <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 390 <212> DNA <213> Artificial Sequence <220> <223> aux promoter gene sequence <400> 1 ttcgttgtgc tgagggaact gagatagatc tcgccagaga aacgttcaat gatttttgct 60 tggagtgaaa aaggcaaata attatagagg aaggaagtca gaaatgctgc gcagtagggc 120 cacttgtata agtgccggtc gaacactgct ggtggaaagt caaaagcgtg aagtattagt 180 tgaactctgt tactaaattg agataaatgg gatattttat tcgaaagtac tgtttgagat 240 ctagcgacaa taataatgtc atcttatgag attgcatggc aatatggatc taatatttgg 300 cataaataga tggtggtttt gtctccactt ttaaaccttc acagcgttac cctaacacct 360 cttaattgcg tacactcctt tcaaccgcat 390 <210> 2 <211> 590 <212> DNA <213> Artificial Sequence <220> <223> tzs gene seqeunce <400> 2 atctcatcta cggaccgact tgcagcggca aaacggacat gcgatccaaa aaccgggtgg 60 ccggtggttg cccttgatcg tgtgcaatgt gtcctcaagt agcggaagac ctttggaatc 120 ggaattgcaa tcaacgcgag aatatatgcc ccctcaccga gggcatcctt gacgctgaga 180 gtgcccacgt cgactctgga ttggcggaag tccgaagacg gtcttattct cgaggggggt 240 cgattattgc atggctaaaa gtccgttttg gagatcgggt tttcatggca tgtcgtcttg 300 gggattcgga cgcctttctc acccgagcca agcacgcgtt gcgccatccg ggaagatcgc 360 ccctcgttgt tggaggagtt ggcgaactct ggccgctcga ccgattttgg aagatatcga 420 cggatatcgc tggcaattcg taacacgatc tcgcaatcag ccagttgcca aatattgatg 480 cgggcggcac tagaggccat agctaatgaa tatcttgaac atgcgctctc caggagcgca 540 gtggccagaa gatggcgcag gacagcctgt ttgcccggta cgctgacgga 590 <110> Korea Forest Research Institute (KFRI) <120> The transformation method of Liriodendron tulipifera embryonic tissue <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 390 <212> DNA <213> Artificial Sequence <220> <223> aux promoter gene sequence <400> 1 ttcgttgtgc tgagggaact gagatagatc tcgccagaga aacgttcaat gatttttgct 60 tggagtgaaa aaggcaaata attatagagg aaggaagtca gaaatgctgc gcagtagggc 120 cacttgtata agtgccggtc gaacactgct ggtggaaagt caaaagcgtg aagtattagt 180 tgaactctgt tactaaattg agataaatgg gatattttat tcgaaagtac tgtttgagat 240 ctagcgacaa taataatgtc atcttatgag attgcatggc aatatggatc taatatttgg 300 cataaataga tggtggtttt gtctccactt ttaaaccttc acagcgttac cctaacacct 360 cttaattgcg tacactcctt tcaaccgcat 390 <210> 2 <211> 590 <212> DNA <213> Artificial Sequence <220> <223> tzs gene seqeunce <400> 2 atctcatcta cggaccgact tgcagcggca aaacggacat gcgatccaaa aaccgggtgg 60 ccggtggttg cccttgatcg tgtgcaatgt gtcctcaagt agcggaagac ctttggaatc 120 ggaattgcaa tcaacgcgag aatatatgcc ccctcaccga gggcatcctt gacgctgaga 180 gtgcccacgt cgactctgga ttggcggaag tccgaagacg gtcttattct cgaggggggt 240 cgattattgc atggctaaaa gtccgttttg gagatcgggt tttcatggca tgtcgtcttg 300 gggattcgga cgcctttctc acccgagcca agcacgcgtt gcgccatccg ggaagatcgc 360 ccctcgttgt tggaggagtt ggcgaactct ggccgctcga ccgattttgg aagatatcga 420 cggatatcgc tggcaattcg taacacgatc tcgcaatcag ccagttgcca aatattgatg 480 cgggcggcac tagaggccat agctaatgaa tatcttgaac atgcgctctc caggagcgca 540 gtggccagaa gatggcgcag gacagcctgt ttgcccggta cgctgacgga 590
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 제3단계; 및
(4) 상기 제3단계로 부터 수득한 체세포배에 geneticin 및 cefotaxime을 처리하여 발아식물체를 재분화하는 제4단계; 를 포함하는 백합나무 배발생조직의 형질전환 방법.
(1) a first step of embryogenesis (embryogenic) tissue Agrobacterium (Agrobacterium) and the air conditioning culture (co-cultivation), the lily trees (Liriodendron tulipifera);
(2) a second step of selecting a transgenic embryonic tissue by treating genetic embryo and cefotaxime to the embryogenic tissue of the co-cultured lily tree obtained from the first step;
(3) a third step of inducing somatic embryos by treating genetic embryos and cefotaxime to the transgenic embryonic tissue obtained from the second step; And
(4) a fourth step of regenerating the germinated plant by treating the genetic cell and cefotaxime to the somatic embryo obtained from the third step; Transformation method of the lily tree embryogenic tissue comprising a.
The Agrobacterium strain according to claim 1, wherein the Agrobacterium is transformed into an Agrobacterium LBA 4404 by introducing an expression vector pBI121 consisting of an aux promoter of SEQ ID NO. Transformation method of lily tree embryogenic tissue, characterized in that the bacterium.
The method of claim 1, wherein the co-culture is 71 to 73 hours.
The method of claim 1, wherein the second step of geneticin and cefotaxime treatment for selection of transformed embryogenic tissue comprises: (a) a step of treating 30 mg / L of geneticin and 250 mg / L cefotaxime for 2 weeks; (b) step b of 50 mg / L geneticin and 250 mg / L cefotaxime for 2 weeks; And (c) treating step 70c / L with geneticin and 250mg / L cefotaxime for 4-6 weeks; Transformation method of the lily tree embryogenic tissue, characterized in that it comprises a.
A lily tree embryogenic tissue transformed by the method of any one of claims 1 to 6.
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| CN110073978A (en) * | 2019-05-24 | 2019-08-02 | 南京林业大学 | It is a kind of using bud as the quick-breeding method of the U.S. Liriodendron chinense in Asia of explant |
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| KR20160066375A (en) | 2014-12-02 | 2016-06-10 | 충북대학교 산학협력단 | Human Neural Stem Cells Expressing Prodrug Conversion Enzyme and Pharmaceutical Composition Comprising the Same for Treating Primary and Metastatic Cancer |
| CN110073978A (en) * | 2019-05-24 | 2019-08-02 | 南京林业大学 | It is a kind of using bud as the quick-breeding method of the U.S. Liriodendron chinense in Asia of explant |
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