KR100788290B1 - Method for mass propagation of oplopanax elatus nakai using seed-derived somatic embryo - Google Patents
Method for mass propagation of oplopanax elatus nakai using seed-derived somatic embryo Download PDFInfo
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Abstract
Description
도 1은 땃두릅나무 종자의 미성숙배(왼쪽)와 층적저장시켜 성숙배로 유기한 성숙배(오른쪽)를 나타낸 것이고,Figure 1 shows the immature embryos (left) and the mature embryos (right) organically stored by stratification of the seedlings of the elder tree,
도 2는 본 발명의 방법에 의하여 배발생 캘러스를 2,4-D를 포함된 배지에서 캘러스의 증식량을 나타낸 것이고,Figure 2 shows the growth rate of callus in the medium containing
도 3은 본 발명의 배발생세포를 무기염 농도를 달리한 배지에서 현탁배양하여 유기된 식물체를 나타낸 것이고,Figure 3 shows the plant grown by suspension culture of embryogenic cells of the present invention in a medium of different inorganic salt concentration,
도 4는 본 발명의 배발생세포를 GA3가 포함된 배지에서 현탁배양하여 유기된 식물체를 나타낸 것이고, 4 is a diagram showing a plant grown by suspension culture of the embryogenic cells of the present invention in a medium containing GA 3 ,
도 5는 본 발명의 배발생세포를 폴리아민이 포함된 배지에서 현탁배양하여 유기된 식물체를 나타낸 것이고, Figure 5 shows the plant grown by suspension culture of the embryogenic cells of the present invention in a medium containing a polyamine,
도 6은 본 발명의 pH를 달리하여 배발생세포를 현탁배양하여 유기된 신초형성율을 나타낸 것이고, Figure 6 shows the induced shoot formation rate by suspension culture of embryogenic cells by varying the pH of the present invention,
도 7은 본 발명의 발아한 체세포배를 고체배지에 배양하여 얻은 유식물체의 수크로 오스 농도에 따른 재분화 양상을 나타낸 것이고,Figure 7 shows the re-differentiation according to the sucrose concentration of the seedlings obtained by culturing the germinated somatic embryo of the present invention in a solid medium,
도 8은 본 발명의 토양 순화 과정을 거쳐 재분화된 식물체를 토양에 이식한 다음 생존한 식물체를 나타낸 것이고,Figure 8 shows the plants survived after transplanting the plants re-differentiated through the soil purification process of the present invention,
도 9는 본 발명의 토양 순화 과정을 거쳐 재분화된 식물체를 토양에 이식한 다음 생존한 식물체의 지상부 생육특성을 나타낸 것이고,9 is a view showing the growth characteristics of the ground portion of the surviving plants after transplanting the plants re-differentiated through the soil purification process of the present invention,
도 10은 본 발명의 토양 순화 과정을 거쳐 재분화된 식물체를 토양에 이식한 다음 생존한 식물체의 지하부 생육특성을 나타낸 것이다. Figure 10 shows the underground growth characteristics of plants that survived after transplanting plants re-differentiated through the soil purification process of the present invention in the soil.
본 발명은 종자 유래의 접합자배에서 유도된 체세포배를 이용한 땃두릅나무 묘목의 대량 생산방법에 관한 것으로, 보다 상세하게는 땃두릅나무 종자를 발아시켜 유도된 접합자배를 고체배지 또는 액체배지에서 배양하여 체세포배를 유기하고 이를 대량 증식시킨 다음, 이로부터 식물체를 재분화하여 토양에 이식시켜 번식시키는 땃두릅나무 묘목의 대량 생산방법에 관한 것이다.The present invention relates to a mass production method of seedling seedlings using somatic embryos derived from seed-derived seedling embryos. More specifically, the seedling embryos induced by germinating seedling seedlings are cultured in solid medium or liquid medium somatic cells. The present invention relates to a method for mass production of seedling tree seedlings, in which pears are organically grown and then grown, and then the plants are regenerated and transplanted into soil.
땃두릅나무(Oplopanax elatus Nakai)는 두릅나무과 땃두릅나무속에 속하는 다년생 낙엽활엽관목이다. 그 분포는 아주 좁은 범위 내에 있는데 전세계적으로 한국, 중국, 러시아 등 3개 국경 내에 소량으로 분포하는 것으로 알려져 있다(Wang et al., 1989). 땃두릅나무는 주로 건조된 뿌리와 줄기를 약으로 사용하고 있는데 뿌리와 줄기에 많은 사포닌, 휘발성 오일, 플라보논, 안트라퀴논, 지방산 등의 성분이 함유되어 있다는 보고가 있으며, 최근 잎에도 많은 유효성분이 함유되어 있다는 보고가 있었는데 이는 뿌리와 줄기의 성분과 큰 차이가 없는 것으로 알려져 있다(Wang et al., 2004).Oplopanax elatus Nakai is a perennial deciduous broad-leaved shrub belonging to the genus Elm and Elm. The distribution is in a very narrow range and is known to be distributed in small amounts within three borders of Korea, China, and Russia (Wang et al., 1989). The elm tree is mainly used for dried roots and stems, and it has been reported that the roots and stems contain many saponins, volatile oils, flavonones, anthraquinones and fatty acids. It is reported that there is no significant difference between the components of root and stem (Wang et al., 2004).
땃두릅나무는 인삼과 비슷한 작용을 하는 것으로 알려져 한약명은 자인삼(刺人蔘)이라 하여 주로 기를 돕고, 심혈관질환, 당뇨병, 풍습 등을 치료하고, 중추신경을 흥분시키는 작용, 신경쇠약, 저혈압, 정신분열증 등에 효과적인 것으로 나타났으며(Zhang et al., 1994), 휘발성 오일은 경련길항작용, 항노화작용, 항염증작용, 항균작용 등에 효능이 있는 것으로 알려져 있다. 인삼, 영지버섯, 마늘 등에 미량으로 들어있는 것으로 알려진 유기게르마늄이 땃두릅나무에서 추출되었는데, 이는 암, 만성성인병 등 난치병으로 알려진 병에 놀라운 효과를 나타내는 것을 일본 의과대학의 임상실험 연구보고에 의해 알려지기 시작하였다(이, 2004).It is known that ginseng tree is similar to ginseng, and the name of Chinese medicine is Jain ginseng (刺 人蔘), which is mainly used to help cure cardiovascular diseases, diabetes, customs, and to stimulate the central nervous system, nervous breakdown, hypotension, mental It has been shown to be effective in schizophrenia (Zhang et al., 1994), and volatile oils are known to be effective in convulsive antagonism, anti-aging, anti-inflammatory and antibacterial activity. Organic germanium, known to contain trace amounts of ginseng, ganoderma lucidum, and garlic, was extracted from the cedar, which has been shown to have remarkable effects on diseases known as incurable diseases such as cancer and chronic adult diseases. Began (2004).
이러한 땃두릅나무는 종자 결실율이 낮고, 종자를 얻었다 하더라도 그 종자의 발아가 아주 곤란하기에 일반적으로 분주, 휘묻이, 꺽꽂이 등 무성생식방법을 사용하는 것으로 알려져 있다(Wang et al., 1989). 그러나 무성생식에서는 미생물 등의 감염으로 인한 불화합성, 대목과 접목의 불활착성, 재료로 사용된 접목의 연령에 따른 차이 등 종합적인 요인이 작용함으로써 그 번식이 어려워지고 분포지역이 좁고 생태군락이 작은 등 단점이 있다. 게다가 현재 자생 땃두릅나무는 인위적인 무차별 남획으로 인해 멸종위기에 처해 있으며, 한국, 중국, 러시아 등 각국에서 2급 보호수로 지정하여 관리하고 있다. It is known to use asexual reproductive methods such as dispensing, burying, and folding because the seedlings have a low seed loss rate and even seed germination is very difficult (Wang et al., 1989). However, in asexual reproduction, comprehensive factors such as incompatibility due to infection of microorganisms, incompatibility of lumber and grafting, and differences according to age of grafting used as materials make the reproduction difficult and distribution area narrow and ecological colony There is a small back disadvantage. In addition, native wild elm tree is endangered by artificial indiscriminate overfishing, and is designated and managed as a second-class protector in Korea, China and Russia.
이에 본 발명자들은 종자를 얻기 어려운 땃두릅나무를 번식시키기 위한 연구를 수행한 결과, 땃두릅나무 종자 유래의 접합자배에서 유도된 체세포배를 이용하면 이를 대량 번식시킬 수 있음을 확인하고 본 발명을 완성하기에 이르렀다.Therefore, the present inventors conducted a study for breeding the elm tree, which is difficult to obtain seeds, and confirmed that the somatic embryo derived from the spliced embryo derived from the elm tree seed was able to reproduce in large quantities and to complete the present invention. Reached.
본 발명은 상기와 같은 요구에 의해 안출된 것으로서, 본 발명의 목적은 멸종위기 식물종인 땃두릅나무 묘목의 기내 대량 생산체계를 확립함으로써 유용한 약리효과를 지니고 있으나 번식이 어려운 땃두릅나무의 대량 생산 방법을 제공하고자 하는 것이다.The present invention has been made in accordance with the above-described demands, and an object of the present invention is to provide a method for mass production of elm elm, which has useful pharmacological effects but is difficult to breed by establishing an in-flight mass production system of endangered elm seedlings. I would like to.
이와 같은 목적을 달성하기 위한 본 발명은 종자 유래의 접합자배에서 유도된 체세포배를 이용한 땃두릅나무 묘목의 대량 생산방법에 관한 것으로 하기의 단계를 포함하여 이루어진 것을 특징으로 한다.The present invention for achieving the above object relates to a mass production method of seedling seedlings using somatic embryos derived from seed germ embryo, characterized in that it comprises the following steps.
⑴ 수세과정을 거쳐 층적저장한 땃두릅나무 종자를 SH 배지 또는 B5 배지에 치상하여 발아시키는 단계와; 치 germinating the seedlings of the elm tree seedlings stratified through washing with SH medium or B5 medium;
⑵ 상기 발아된 기내 식물체의 절편체를 액체배지 또는 고체배지에 치상하고 배양하여 체세포배를 유도시키는 단계와; 치 inducing somatic embryos by densifying and incubating the germinated explants of the plant in a liquid medium or a solid medium;
⑶ 상기 유도된 체세포배를 액체배지 또는 고체배지에서 배양하여 증식시키는 단계 와; 증식 culturing the induced somatic cell culture in a liquid medium or a solid medium;
⑷ 상기 증식된 체세포배를 액체배지 또는 고체배지에서 배양하여 유식물체를 재분화시키는 단계와;배양 re-differentiating seedlings by culturing the proliferated somatic cell medium in a liquid medium or a solid medium;
⑸ 상기 재분화된 유식물체를 순화시키는 단계.를 purifying the regenerated seedlings.
본 발명의 상기 제 ⑴단계에서의 배지는 수크로오스가 3%(w/v) 수준으로 첨가된 SH 배지 또는 B5 배지인 것을 특징으로 하며, 상기 발아는 25℃의 16시간 광조건과 8시간 암조건에서 수행되는 것을 특징으로 한다.The medium of the first step of the present invention is characterized in that the sucrose is added to 3% (w / v) level of the SH medium or B5 medium, the germination at 25 ℃ 16 hours light conditions and 8 hours dark conditions Characterized in that it is carried out.
본 발명의 상기 제 ⑵단계에서의 배지는 상기 ⑵ 단계에서의 배지는 SH 배지 또는 B5 배지인 것을 특징으로 하며, 상기 배양은 25℃의 16시간 광조건과 8시간 암조건에서 수행되는 것을 특징으로 한다.The medium in the step iii of the present invention is characterized in that the medium in the ⑵ step is SH medium or B5 medium, the culture is characterized in that carried out at 25 ℃ 16 hours light conditions and 8 hours dark conditions. .
본 발명의 상기 제 ⑶단계에서의 배지는 1/3 MS 배지 또는 1/4 MS 배지인 것을 특징으로 하며, 상기 배양은 25℃의 16시간 광조건과 8시간 암조건에서 수행되는 것을 특징으로 한다.The medium in the first step of the present invention is characterized in that 1/3 MS medium or 1/4 MS medium, the culture is characterized in that it is carried out at 25 ℃ 16 hours light conditions and 8 hours dark conditions.
본 발명의 상기 제 ⑷단계에서의 배지는 수크로오스가 1% 내지 3%(w/v) 수준으로 첨가된 1/3 MS 배지 또는 1/4 MS 배지인 것을 특징으로 하며, 상기 배양은 25℃의 16시간 광조건과 8시간 암조건에서 수행되는 것을 특징으로 한다.The medium in step iii of the present invention is characterized in that 1/3 MS medium or 1/4 MS medium added with sucrose at a level of 1% to 3% (w / v), the culture is 25 ℃ It is characterized in that it is carried out in 16 hours light conditions and 8 hours dark conditions.
또한, 본 발명의 상기 제 ⑷단계에서의 배지의 pH는 5.0 내지 5.75 범위인 것을 특징으로 한다.In addition, the pH of the medium in the first step of the present invention is characterized in that the range of 5.0 to 5.75.
본 발명의 상기 ⑸ 단계에서의 순화는 상토와 모래가 1：1로 혼합 조성된 토 양에서 수행되는 것을 특징으로 한다.Purification in the step (i) of the present invention is characterized in that it is carried out in the soil mixed with topsoil and sand 1: 1.
이하, 본 발명의 구성을 바람직한 실시예를 통하여 보다 상세히 설명할 것이나, 이들 실시예는 오로지 본 발명을 구체적으로 예시하기 위한 것으로서 본 발명의 범위가 이들 실시예에 의해 제한되는 것은 아니며, 본 발명의 범위는 오직 특허청구범위에 기재된 바에 의해 한정되어야 할 것이다.Hereinafter, the configuration of the present invention will be described in more detail with reference to preferred embodiments, but these embodiments are only for illustrating the present invention specifically, the scope of the present invention is not limited by these examples, The scope shall be defined only by what is stated in the claims.
<실시예 1> 종자 발아 및 체세포배 유도Example 1 Seed Germination and Somatic Embryogenesis
땃두릅나무 종자는 다른 일반 식물체들과는 달리, 먼저 종자로부터 성숙한 배가 밖으로 노출된 상태에서 지속적인 배양을 하면 뿌리가 출현하게 되고, 이어서 자엽의 개엽 및 식물체의 생장이 이루어지는 것으로 발아과정이 진행되는데, 본 발명에서 층적저장된 종자를 이용하는 것은 땃두릅나무 종자의 미숙 배를 성숙 배로 효과적으로 전환시키기 위함이다. 층적저장방법은 땃두릅나무 종자를 15℃에서 5개월 처리한 후 4℃에서 2개월동안 처리하였다(도 1 참조).Unlike other general plants, the seedlings of young elm tree are first sprouted by continuous cultivation with the mature pears exposed from the outside, and then the germination process proceeds as the cotyledons and the growth of the plants are carried out. The use of layered seeds is to effectively convert the immature pears of the seedwood to mature pears. The layered storage method was treated for 5 months at 15 ° C. seedlings for 2 months at 4 ° C (see Figure 1).
수세과정을 거쳐 층적저장된 땃두릅나무 (Oplopanax elatus Nakai) 종자를 수돗물에 세척한 다음 10% 상업용 락스 용액에 20분간 표면살균을 하였다. 표면살균된 종자를 무균작업대 내에서 멸균수로 3~4회 세척한 다음, 멸균된 여과지에서 표면의 수분을 제거하였다.Structural Stolled Elm ( Olopanax) elatus Nakai) seeds were washed in tap water and then surface sterilized for 10 minutes in a 10% commercial Lax solution. The surface sterilized seeds were washed 3 to 4 times with sterile water in a sterile workbench, and then surface moisture was removed from the sterilized filter paper.
표면살균된 땃두릅나무 종자를 각각 1/2 MS(Murashige and Skoog) 배지, 1/4 MS 배지, SH(Schenck and Hildebrandt) 배지, B5 배지에 치상하여 6주 후 발아율을 조사하였다. 또한, 1/2 MS 배지 및 1/4 MS 배지에 2,4-D 1.0㎎/ℓ의 생장조절제가 첨가된 배지를 조성하였다. 한편, 각 배지의 수크로오스 농도는 3%(w/v)로 조성하였다. 종자발아는 25℃에서 16시간의 광조건과 8시간의 암조건으로 배양하였고, 각 페트리디쉬(100×40mm)에 10개씩 치상하였고, 6반복 처리하였다.Surface sterilized seedlings were seeded on 1/2 MS (Murashige and Skoog) medium, 1/4 MS medium, Schenck and Hildebrandt (SH) medium, and B5 medium, respectively, and germination rates were examined after 6 weeks. In addition, a medium in which 2,4-D 1.0 mg / L growth regulator was added to 1/2 MS medium and 1/4 MS medium was prepared. Meanwhile, the sucrose concentration of each medium was 3% (w / v). Seed germination was incubated at 25 ° C. under 16 hours of light conditions and 8 hours of dark conditions. Ten seedlings were placed on each Petri dish (100 × 40 mm) and treated with 6 repetitions.
그 결과, SH 배지가 종자 발아율에서 52.4%로 가장 효과적이었으며, B5 배지에서도 실용적인 수준인 43.6%로 높게 나타났으며, 1/2 MS 배지 및 1/4 MS 배지에서는 발아율이 낮은 것으로 관찰되었다 (표 1).As a result, SH medium was the most effective at 52.4% in seed germination rate, 43.6% was high in B5 medium, and low germination rate was observed in 1/2 MS and 1/4 MS medium. One).
상기 발아된 기내 식물체를 0.5㎠ 크기로 잘라낸 절편체를 상기와 같은 동일 배지 조성에서 치상하고 배양하여 6주간 배양하여 체세포배 형성율을 조사하였다. 25℃에서 16시간의 광조건과 8시간의 암조건으로 배양한 결과, 체세포배 형성율은 SH 배지, B5 배지, 생장조절제를 첨가하지 않은 1/2 MS 배지, 생장조절제를 첨가하지 않은 1/4 MS 배지에서 비슷한 수준인 것으로 나타났다. 결과적으로 종자 발아율 및 체세포형성율을 감안할 때, 땃두릅나무 종자 유래의 체세포배 유도에 바람직한 배양배지는 SH 배지 또는 B5 배지인 것으로 나타났다.The germinated explants were cut into 0.5
[표 1] 배지 종류 및 염 농도가 땃두릅나무 종자의 발아율 및 체세포배 형성에 미 치는 영향[Table 1] Effect of Medium Type and Salt Concentration on Germination Rate and Somatic Embryogenesis of Seed elm
배발생캘러스의 증식을 위하여 auxin의 일종인 2,4-D를 1㎎/ℓ, 2㎎/ℓ, 3㎎/ℓ, 4㎎/ℓ의 농도로 사용하여 실험을 진행하였다. Auxin은 세포분열을 촉진하는 작용을 하므로 본 실험에서 배지 중에서 쉽게 분해되지 않고 오랫동안 유지되는 특성을 갖고 있고, 일상적으로 많이 사용되는 2,4-D를 선택하여 실험에 사용하였다. Callus의 증식은 1/2 MS 배지에 2,4-D 1㎎/ℓ가 첨가된 배지에서 가장 효과적인 것으로 나타났으며, 다음으로는 1/2 MS 배지에 2,4-D 2㎎/ℓ을 첨가된 배지에서 효과적인 것으로 나타났으며, 생장조절물질이 첨가되지 않은 1/2 MS 배지에서는 callus가 증식되지 않았다(도 2 참조). 이렇게 2,4-D가 첨가된 배지에서 증식시킨 배발생캘러스를 호르몬이 없는 배지에 옮겨 성숙상태의 체세포배로 유도한다.For the growth of embryogenic callus, the experiment was performed using 2,4-D, a kind of auxin, at concentrations of 1 mg / l, 2 mg / l, 3 mg / l, and 4 mg / l. Auxin has a characteristic of promoting cell division, so it is not easily decomposed in the medium in this experiment and has a long-lasting property, and 2,4-D, which is commonly used in daily life, was selected and used in the experiment. Proliferation of Callus was most effective in medium supplemented with 2,4-
<실시예 2> 체세포배로부터 식물체 재분화Example 2 Plant Regeneration from Somatic Embryos
상기 실시예 1에서 수득된 땃두릅나무 종자 유래 체세포배를 1/2 MS 배지, 1/3 MS 배지, 1/4 MS 액체배지 각각에 1%(w/v) 수크로오스를 첨가한 배지가 각각 50㎖씩 분주된 250㎖ 삼각플라스크에 각각 치상하여, shaker에 100rpm으로 조절하여 25℃에서 16시간의 광조건과 8시간의 암조건 조건에서 증식시켰다.50 ml of the medium in which 1% (w / v) sucrose was added to 1/2 MS medium, 1/3 MS medium, and 1/4 MS liquid medium, respectively, was used to obtain the somatic embryo derived from Sesame seedwood obtained in Example 1 Each of them was placed in a 250 ml Erlenmeyer flask, which was then dispensed, and the shaker was adjusted to 100 rpm to grow at 25 ° C. under 16 hours of light conditions and 8 hours of dark conditions.
그 결과, 땃두릅나무의 체세포배의 증식은 1/3 MS 배지에서 가장 양호한 결과를 보였으며, 신초(shoot) 형성도 100%로 가장 양호한 것으로 나타났으며, 다음으로 1/4 MS 배지가 양호하였으며, 1/2 MS 배지에서는 효율이 낮은 것으로 나타났다 (표 2).As a result, the proliferation of somatic embryos of elder tree was the best in 1/3 MS medium, shoot formation was also best at 100%, and the next 1/4 MS medium was good. , Efficiency was low in 1/2 MS medium (Table 2).
체세포배의 증식이 1/4 MS 배지에서 양호한 결과를 나타낸 것은 식물체에서의 체세포배 증식은 대부분은 스트레스로 인하여 형성되는 것으로 알려져 있는데, 이는 아마도 낮은 염 농도에 의한 양분 스트레스에 기인한 것으로 여겨지며, 신초 형성은 식물체가 형성되는데 필요한 양분의 결핍으로 인하여 1/4 MS 배지에서의 신초 형성율이 상대적으로 염 농도가 높은 두 배지에 비하여 낮게 나타난 것으로 여겨진다.Proliferation of somatic embryos showed good results in 1/4 MS medium. Somatic embryo proliferation in plants is known to be formed largely due to stress, presumably due to nutrient stress due to low salt concentrations. Formation is believed to result in a lower rate of shoot formation in 1/4 MS media compared to two media with high salt concentrations due to the lack of nutrients needed for plant formation.
[표 2] 배지 조성에 따른 땃두릅나무 종자 유래 체세포배의 증식 및 재분화[Table 2] Proliferation and Regeneration of Somatic Embryos from Aspergillus japonica Seed according to Medium Composition
한편, 액체배양은 고체배양과 그 생장조건이 다를 수 있기 때문에 최적 배양 조건을 확립하기 위하여 1/3 MS 액체배지와 1/3 MS 고체 배지로 배지 형태만을 달리하여 상기와 동일한 배양 조건에서 배양하였다. 그 결과 신초 형성이 액체배지에서 더욱 효과적으로 이루어짐을 알 수 있었고, 반면 고체배지에서는 액체배지에 비하여 식물체의 유리화 현상(vitrification)이 거의 발생하지 않는 것으로 나타났다. 또한, 유리화된 식물체로부터 정상적인 식물체로의 전환은 액체배지에서 형성된 유리화 신초를 고체배지에 계대배양함으로써 정상적인 식물체로의 전개가 이루어지는 것을 관찰할 수 있었다 (표 3).On the other hand, the liquid culture is different from the solid culture and its growth conditions may be different, so in order to establish the optimum culture conditions were cultured in the same culture conditions by changing only the medium type with 1/3 MS liquid medium and 1/3 MS solid medium. . As a result, it was found that shoot formation was more effective in the liquid medium, whereas the vitrification of the plants hardly occurred in the solid medium as compared to the liquid medium. In addition, the conversion from the vitrified plant to the normal plant was observed by the passage of vitrified shoots formed in the liquid medium to the solid medium to develop the normal plant (Table 3).
[표 3] 배지 형태에 따른 땃두릅나무 종자 유래 체세포배의 증식 및 재분화Table 3 Proliferation and Regeneration of Somatic Embryos Derived from Sesame Seeds According to Medium Type
효과적인 식물체로의 재분화율을 알아보기 위하여 체세포배를 60메쉬의 체로 선별한 배발생 세포를 MS 배지, 1/2 MS 배지, 1/3 MS 배지, 1/4 MS 배지, 1/8 MS 배지 각각에 1%(w/v) 수크로오스를 첨가한 배지가 각각 100㎖씩 분주된 250㎖ 삼각플라스크에 플라스크당 각각 20㎎ 수준으로 접종하여, shaker에 135rpm으로 조절하여 25℃에서 16시간의 광조건과 8시간의 암조건으로 배양한 결과, 접종 4주후에 체세포배의 발아가 시작되어 8주후에 체세포배의 완전한 성숙 및 식물체로의 재분화 가 확인되었다. 또한, MS 배지에서는 2차배의 발달과 성숙이 관찰되었으며, 1/2 MS 배지 및 1/3 MS 배지에서는 거의 발아하는 것을 확인할 수 있었다(도 3 참조).To determine the effective rate of regeneration into plants, embryonic cells selected from 60 mesh sieves were selected from MS medium, 1/2 MS medium, 1/3 MS medium, 1/4 MS medium, and 1/8 MS medium, respectively. 100 ml of 1% (w / v) sucrose was added to a 250 ml Erlenmeyer flask, each of which was dispensed with 100 ml each, at a 20 mg level per flask, and adjusted to 135 rpm in a shaker. As a result of incubation under dark conditions of time, germination of somatic embryos started at 4 weeks after inoculation and complete maturation and regeneration into plants at 8 weeks. In addition, the development and maturation of the secondary fold was observed in MS medium, it was confirmed that the germination almost in 1/2 MS medium and 1/3 MS medium (see Fig. 3).
체세포배의 발아에 있어서 polyamine류와 GA3의 영향을 평가하기 위하여 1/3 MS 배지에 GA3, spermidine, spermine을 농도별로 처리한 결과, GA3는 0.01㎎/ℓ, 0.1㎎/ℓ, 2㎎/ℓ의 모든 처리 농도에서 60메쉬 이하의 배발생세포 접종후 3주만에 체세포배의 발아가 활발하게 시작되는 것을 관찰할 수 있었고(도 4 참조), 접종 3주후 polyamine류에서 putrescine은 체세포배의 발달, 성숙을 관찰할 수 없었으며, spermidine은 0.01㎎/ℓ에서, spermine은 2㎎/ℓ에서 체세포배로의 발달과 발아를 관찰할 수 있었다(도 5 참조). 따라서, 바람직하기로는 1/3 MS 배지에 GA3를 처리한 배지가 체세포배의 발아에 효과적인 것으로 평가되었다.After a treatment on 1/3 MS medium by GA 3, spermidine, spermine concentrations to assess the effects of GA 3 and polyamine acids in the germination of somatic embryos, GA 3 is 0.01㎎ / ℓ, 0.1㎎ / ℓ, 2 It was observed that germination of somatic embryos started actively 3 weeks after inoculation of embryonic cells of 60 mesh or less at all treatment concentrations of ㎎ / l (see FIG. 4), and putrescine was somatic embryos in polyamines after 3 weeks of inoculation. The development and maturation of the spermidine could not be observed, and the development and germination of spermidine at 0.01 mg / l and spermine at 2 mg / l could be observed (see FIG. 5). Therefore, preferably, a medium treated with GA 3 in 1/3 MS medium was evaluated to be effective for germination of somatic embryos.
배지의 pH는 생장조절제와 기타 양분의 흡수뿐만 아니라 교질의 특성을 변화시켜서 수분의 이용성에도 작용하여 결국은 배양체의 분화와 발달에 영향을 미친다. 배양배지의 pH를 달리하여 신초 형성율을 조사한 결과, pH 5.75에서 80%로 가장 양호한 신초 형성율을 보였고, 다음으로 pH 5와 pH 7에서 40% 수준의 비교적 양호한 신초 형성율을 나타냈으며, pH 4와 pH 6에서는 신초 형성율이 실용적이지 못한 것으로 나타났다 (도 6 참조). 결과적으로 바람직한 배지의 pH 범위는 5.0 내지 5.75인 것으로 평가되었다.The pH of the medium not only absorbs growth regulators and other nutrients, but also affects the availability of water by changing the properties of the colloids, which in turn affects the differentiation and development of the culture. As a result of investigating the shoot formation rate by changing the pH of the culture medium, it showed the best shoot formation rate at 80% at pH 5.75, and showed a relatively good shoot formation rate at 40% at
액체배지에서 발아된 체세포배의 고체배지에서의 적응 정도, 즉 MS 농도에 따른 식물체 재분화 양상은 처음 한달 동안에는 1/8 MS 배지에서 뿌리 및 신초가 모두 형성되었으나, 3달 후 그 신장이 거의 이루어지지 않았으며, 원뿌리로의 분화가 없고 곁뿌리 형태의 뿌리만을 가지고 있어 재분화 배지로 적합하지 않은 것으로 평가되었다. 한편 1/3 MS 배지와 1/4 MS 배지에서는 신초 형성 후 뿌리 형성이 활발하게 이루어져 원뿌리를 형성하는 것이 관찰되어 재분화를 위한 바람직한 배지로 평가되었다 (표 4 및 표 5).The degree of adaptation of the somatic cell germination in liquid medium, ie, plant regeneration according to MS concentration, formed both roots and shoots in 1/8 MS medium during the first month, but almost no elongation after 3 months. No root differentiation and only roots in the form of side roots were considered to be unsuitable for regeneration medium. Meanwhile, in the 1/3 MS medium and the 1/4 MS medium, root formation was actively formed after shoot formation to form a root root, which was evaluated as a preferable medium for regeneration (Table 4 and Table 5).
[표 4] MS 배지 조성에 따른 식물체 재분화 양상 (배양 1달 후)Table 4 Plant Regeneration Patterns According to MS Medium Composition (After 1 Month of Culture)
* Each value is the mean of at least three independent experiments ± SD. * Each value is the mean of at least three independent experiments ± SD.
† ; Within columns, means followed by the same letters are not significantly different at ρ=0.05 according to Duncan's multiple range test. † ; Within columns, means followed by the same letters are not significantly different at ρ = 0.05 according to Duncan's multiple range test.
[표 5] MS 배지 조성에 따른 식물체 재분화 양상 (배양 3달 후)Table 5 Plant Regeneration Patterns According to MS Medium Composition (After 3 Months of Culture)
† ; Each value represents the mean±S.D. † ; Each value represents the mean ± SD
‡ ; Within columns, means followed by the same letters are not significantly different at ρ=0.05 according to Duncan's multiple range test. ‡ ; Within columns, means followed by the same letters are not significantly different at ρ = 0.05 according to Duncan's multiple range test.
배지의 수크로오스 농도에 따른 식물체 재분화 양상을 알아보기 위하여 1/3 MS 기본배지에 수크로오스 농도를 달리하여 조성한 배지에 상기와 같이 액체배지에서 체세포배 발생을 통해 성장한 발아단계의 체세포배를 치상하여 재분화 특성을 조사하였다. 배지의 수크로오스 농도(w/v)에 따른 식물체 재분화 양상은 배양 1달 후의 관찰에서 3%에서 활발한 재분화가 관찰되었고, 3달 후에도 비슷한 수준으로 양호한 것으로 나타났다 (도 7 B 참조). 0.5% 수준에서는 처음 2주 정도는 신초 형성이 활발하였으나, 결과적으로 뿌리 형성이 잘되지 않았다 (도 7 C 참조). 5% 수준에서는 거의 백화현상을 일으켰으며(도 7 A 참조), 0% 수준에서는 갈변됨을 관찰 할 수 있었다 (도 7 D 참조). 전체적으로 1% 내지 3%의 수크로오스 농도 범위에서 유식물체로의 재분화가 양호한 것으로 평가되었다 (표 6, 표 7).In order to examine the regeneration patterns of plants according to the sucrose concentration of the medium, the regeneration characteristics of the germination stage grown through the somatic embryo development in the liquid medium in the medium prepared by varying the sucrose concentration in the basic medium of 1/3 MS Was investigated. Plant regeneration according to sucrose concentration (w / v) of the medium showed active regeneration at 3% in observations after 1 month of culture, and similar levels were good after 3 months (see FIG. 7B). At 0.5% level, shoot formation was active for the first two weeks, but as a result, root formation was poor (see FIG. 7C). At 5% level, almost whitening occurred (see FIG. 7A), and browning was observed at 0% level (see FIG. 7D). Overall, differentiation into seedlings in the sucrose concentration range of 1% to 3% was evaluated as good (Table 6, Table 7).
[표 6] 배지의 수크로오스 농도에 따른 식물체 재분화 양상 (배양 1달 후)TABLE 6 Plant Regeneration Patterns According to Sucrose Concentration of Medium (After 1 Month of Culture)
* Each value is the mean of at least three independent experiments ± SD. * Each value is the mean of at least three independent experiments ± SD.
†; Within columns, means followed by the same letters are not significantly different at ρ=0.05 according to Duncan's multiple range test. † ; Within columns, means followed by the same letters are not significantly different at ρ = 0.05 according to Duncan's multiple range test.
[표 7] 배지의 수크로오스 농도에 따른 식물체 재분화 양상 (배양 3달 후)TABLE 7 Plant Regeneration Patterns According to Sucrose Concentration of Medium (After 3 Months of Culture)
† ; Each value represents the mean±S.D. † ; Each value represents the mean ± SD
‡ ; Within columns, means followed by the same letters are not significantly different at ρ=0.05 according to Duncan's multiple range test. ‡ ; Within columns, means followed by the same letters are not significantly different at ρ = 0.05 according to Duncan's multiple range test.
<실시예 3> 유식물체의 순화Example 3 Purification of Seedlings
실시예 2의 액체배지에서 체세포배 발생을 통해 성장한 발아단계의 체세포배를 1/3 MS 기본배지에 1%(w/v)의 수크로오스를 첨가한 고체배지에 옮겨서 3개월간 배양하여 자란 유식물체로 순화를 수행하였다. 2주동안 25℃에서 16시간의 광조건과 8시간의 암조건의 생장상(growth chamber)에서 적응시키고 2주동안 50%차광막이 처리된 온실에 2주동안 생육시킨 결과, 30㎜ 이상의 유식물체에서 100%의 순화율을 보였고, 20㎜ 정도의 유식물체에서는 85%의 비교적 높은 순화율을 보였다. 그러나 식물체의 생존율은 대체로 높은 편이었으나, 순화를 거친 식물체의 상태는 30㎜ 이상의 유식물체의 경우 매우 양호한 반면, 20㎜ 미만의 유식물체의 경우 빈약한 것으로 나타났다 (도 8 참조).Somatic embryos in the germination stage grown through somatic embryo development in the liquid medium of Example 2 were transferred to a solid medium containing 1% (w / v) sucrose in a 1/3 MS basic medium and cultured for 3 months to seedlings. Purification was performed. Adapted in a growth chamber of 16 hours of light conditions and 8 hours of dark conditions at 25 ° C. for 2 weeks, and grown for 2 weeks in a greenhouse treated with 50% shading film for 2 weeks. The purity was 100%, and the relatively high purity was 85% in the 20 mm seedlings. However, the survival rate of the plants was generally high, but the state of the purified plants was very good for the seedlings of 30 mm or more, but poor for the seedlings of less than 20 mm (see Fig. 8).
토양조율에 따른 순화효율성을 알아보기 위하여 기내배양 식물체를 순화 토양의 조성을 달리하여 5개월간 생육시킨 후 초장, 엽폭, 엽장을 조사하고, 발근정도를 뿌리의 지름과 길이를 조사하여 평가하였다. 순화 식물체의 엽폭, 엽장은 비슷한 수준을 나타내었으며, 초장은 상토에 각각 모래와 펄라이트를 조합한 토양과 상토+버미큘라이트+펄라이트를 조합한 토양에서 높은 수준으로 나타났다(도 9 참 조). 순화 식물체의 뿌리의 지름을 보면, 상토+펄라이트를 조합한 토양과 상토+버미큘라이트+펄라이트를 조합한 토양에서 낮은 수준이었고, 상토+모래의 토양 조건에서 뿌리의 지름과 길이로 보아 발근정도가 양호하였다. 이를 종합하여 볼 때, 바람직한 순화 토양의 조성은 상토와 모래를 1：1로 혼합하여 조성하는 것이 가장 효과적인 것으로 평가되었다 (도 10 참조).In order to determine the purifying efficiency according to soil conditioning, in-flight cultured plants were grown for 5 months with different composition of the purified soil, and then the height, leaf width, and leaf length were examined, and rooting degree was evaluated by examining root diameter and length. The leaf width and leaf length of the purified plant showed similar levels, and the grass length was high in the soil with sand and perlite and the soil with vermiculite and perlite, respectively (see Fig. 9). The root diameters of the purified plants were low in soils with top soil + pearlite and soils with top soil + vermiculite + pearlite, and rooting was good in terms of root diameter and length under soil + sand soil conditions. . Taken together, it was evaluated that the most preferable composition of the purified soil was to mix and mix the top soil and sand in a 1: 1 ratio (see FIG. 10).
이상에서 상술한 바와 같이 본 발명에 따른 종자 유래 접합자배에서 유도된 체세포배를 이용한 땃두릅나무 묘목의 대량 생산방법은 멸종위기 식물종인 땃두릅나무 묘목의 효과적인 기내 대량 생산방법인 평가되었다.As described above, the mass production method of the seedling tree seedlings using the somatic embryo derived from the seed-derived zygomatic embryos according to the present invention was evaluated as an effective in-flight mass production method of the seedling seedlings of endangered plant species.
또한, 본 발명의 따른 종자 유래 체세포배를 이용한 땃두릅나무 묘목의 대량 생산방법은 번식이 어려운 땃두릅나무의 효과적인 현지외 보존수단으로 평가되었고, 이를 이용하여 땃두릅나무의 유용한 약리효과를 실용화시킬 수 있는 방법을 제공할 수 있을 것으로 기대되었다.In addition, the method of mass production of seedling seedlings using seed-derived somatic embryos according to the present invention has been evaluated as an effective off-site preservation means for the difficult to breed seedlings. It was expected to be able to provide.
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