KR20150057052A - A composition for improvement of regeneration system from protcorm-like body of Cymbidium plants and method therefor - Google Patents
A composition for improvement of regeneration system from protcorm-like body of Cymbidium plants and method therefor Download PDFInfo
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Abstract
Description
본 발명은 심비디움 원괴체 유사체로부터의 재분화 개선용 조성물 및 그 방법에 관한 것으로, 더욱 상세하게는 심비디움 PLB (원괴체유사체; protocorm-like body)를 증식시킨 후, PLB로부터 신초를 대량 형성시키는 과정에서 비타민 C와 질산은 처리를 통해 신초발생을 촉진시키고 조직배양 과정에서 흔히 발생하는 갈변화를 감소시켜 전반적인 재분화 효율을 향상시키고 또한, 장기간 배양 시 발생하는 변이체 발생을 감소시키는 방법 및 그 조성물에 관한 것이다.
The present invention relates to a composition for improving regeneration from a cymbidium original protozoan analogue and a method thereof, and more particularly to a composition for regenerating cymbidium PLB (protocorm-like body) The present invention relates to a method and composition for promoting shoot formation through vitamin C and nitric acid treatment and reducing the occurrence of brownish discoloration commonly occurring during tissue culture, thereby improving the overall regeneration efficiency and reducing the occurrence of mutants occurring during long-term culture.
일반적으로 심비디움을 포함하는 양란류의 품종개량은 배수체 육종, 교잡육종 및 돌연변이 육종 방법 등이 이용되어 왔으며, 주요 육종목표로는 새로운 화색 창출, 내병성 및 내서성 등 생육이 강건하며 엽의 모양이 특이한 것, 절화수명, 단위면적 당 생산성이 높은 것 그리고 개화시기가 다양한 것, 생산비 절감형 및 상품성 증대 등이 있다. 이러한 육종목표를 만족하는 우수 품종을 개발해도 이러한 품종이 우수 형질을 유지한 상태로 농가재배 및 지역적응성 시험을 거쳐 대량 증식과정을 거쳐 소비자에게 까지 보급되는 효과를 기대할 수 있다. 즉 우수 품종개발도 중요하지만 이를 뒷받침 해줄 우량 형질을 유지하게 하는 대량 증식체계의 확립 없이는 우량 심비디움 품종보급이 어려운 실정이다. 이러한 심비디움을 포함하는 양란 대량증식에 있어서 필수적인 PLB의 증식 및 신초재분화 과정에서 발생하는 갈변화 감소 및 변이체 감소 등의 문제를 해결하는 재분화 체계 도입이 필요한 실정이다.In general, varieties of cymbidium, including cymbidium, have been used for breeding of diploid breeding, hybrid breeding, and mutation breeding. Major breeding targets include the development of new colors, tolerance to disease, , A cut flower life, a high productivity per unit area, a variety of flowering time, a reduction in production cost, and an increase in commerciality. Even if the best varieties satisfying these breeding goals are developed, it can be expected that these varieties will be distributed to the consumers through the mass proliferation process after the farming and local adaptability tests with the excellent traits maintained. In other words, development of excellent varieties is also important, but it is difficult to supply superior Cymbidium varieties without establishing a mass proliferation system that maintains excellent traits to support them. It is necessary to introduce a regeneration system that solves the problems of PLB growth and reduction of brown color change and reduction of variant occurring during the process of shoot regeneration, which are essential for the mass propagation of cyanobacteria including cymbidium.
[관련 특허 문헌][Related Patent Literature]
대한민국 특허공개번호 제1020130052780호Korean Patent Publication No. 1020130052780
본 발명은 상기의 필요성에 의하여 안출된 것으로서 본 발명의 목적은 심비디움 PLB로부터 재분화 효율을 증대시키고 갈변화 및 변이체 발생을 감소시키는 방법을 제공하는 것이다.The present invention has been made in view of the above needs, and an object of the present invention is to provide a method for increasing the regeneration efficiency from Cymbidium PLB and reducing the occurrence of chrysanthemum variants and mutants.
본 발명의 다른 목적은 심비디움 PLB로부터 재분화 효율을 증대시키고 갈변화 및 변이체 발생을 감소시키는 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition that increases the efficiency of regeneration from cymbidium PLB and reduces churning and mutagenesis.
상기의 목적을 달성하기 위하여 본 발명은 심비디움 원괴체유사체(PLB; protocorm-like body)를 증식시킨 후, 상기 PLB 조직으로부터 신초를 형성하여 재분화하는 단계에서 비타민 C 또는 질산은 처리를 수행하는 단계를 포함하는 신초 발생을 촉진시키는 방법을 제공한다.In order to achieve the above object, the present invention includes a step of growing a protocorm-like body (PLB) and then performing vitamin C or nitric acid treatment at the step of regenerating shoots from the PLB tissue Lt; RTI ID = 0.0 > of < / RTI >
본 발명의 일 구현예에 있어서, 상기 비타민 C 농도는 50 mM인 것이 바람직하고, 상기 질산은 농도는 20 mM인 것이 바람직하나 이에 한정되지 아니한다.In one embodiment of the present invention, the vitamin C concentration is preferably 50 mM, and the silver nitrate concentration is preferably 20 mM, but is not limited thereto.
본 발명의 다른 구현예에 있어서, 상기 처리 단계는 질산은과 비타민 C를 동시에 처리하는 것이 바람직하나 이에 한정되지 아니한다.In another embodiment of the present invention, the treatment step is preferably but not limited to treating silver nitrate and vitamin C at the same time.
또 본 발명은 심비디움 원괴체유사체(PLB; protocorm-like body)를 증식시킨 후, 상기 PLB 조직으로부터 신초를 형성하여 재분화하는 단계에서 비타민 C 또는 질산은 처리를 수행하는 단계를 포함하는 신초형성 과정에 나타나는 갈변화 및 변이 식물개체 발생 감소 방법을 제공한다.In addition, the present invention relates to a method for producing glutinous cells, which comprises a step of propagating a protocorm-like body (PLB) and then performing vitamin C or silver nitrate treatment in the step of regenerating shoots from the PLB tissue And a method for reducing the incidence of mutant plants.
또 본 발명은 비타민 C 및 질산은 또는 이들의 혼합물을 유효성분으로 포함하는 심비디움 원괴체유사체(PLB; protocorm-like body)로부터 신초 발생 촉진용 조성물을 제공한다.The present invention also provides a composition for promoting shoot initiation from a protobormium-like body (PLB) comprising vitamin C and silver nitrate or a mixture thereof as an active ingredient.
또 본 발명은 비타민 C 및 질산은 또는 이들의 혼합물을 유효성분으로 포함하는 신초형성 과정에 나타나는 갈변화 현상 감소용 조성물을 제공한다.The present invention also provides a composition for reducing the browning phenomenon appearing in the process of shoot formation comprising vitamin C and silver nitrate or a mixture thereof as an active ingredient.
또 본 발명은 비타민 C 및 질산은 또는 이들의 혼합물을 유효성분으로 포함하는 심비디움 식물체의 조직배양 시 발생하는 변이체 발생 감소용 조성물을 제공한다.The present invention also provides a composition for reducing the occurrence of mutants occurring in tissue culture of a cymbidium plant comprising vitamin C and silver nitrate or a mixture thereof as an active ingredient.
이하 본 발명을 설명한다.Hereinafter, the present invention will be described.
본 발명은 심비디움 PLB로부터 재분화 효율을 증대시키고 갈변화 및 변이체 발생을 감소시키는 방법을 제공하는 것이다. 상세하게는 심비디움 재분화 효율향상 실험에 사용될 절편체로 심비디움의 PLB를 사용하여 우선 갈변화가 최소로 발생하는 효과를 구명하기 위해 비타민 C와 질산은 첨가를 통해 효과를 비교하여 최적의 처리조합을 선정한 후, 역시 동일한 농도로 신초형성율에 비타민 C와 질산은을 처리하여 갈변화도 감소되고 신초 형성촉진을 통한 재분화 효율 향상을 도모하였다. 또한 갈변화 감소방지 및 신초형성 촉진효과가 있는 비타민 C와 질산은을 대상으로 각각 단독 및 혼합처리를 통해 장기간의 심비디움 식물체의 조직배양 시 발생하는 변이체 발생을 감소시켰다The present invention provides a method for increasing the regeneration efficiency from cymbidium PLB and reducing the occurrence of chrysanthemum variants and mutants. In particular, to evaluate the effect of PLB of Cymbidium as a slice to be used in the experiment to improve the regeneration efficiency of cymbidium, the effects of the addition of vitamin C and silver nitrate were compared with each other, In the same concentration, the shoot formation rate was also decreased by treatment with vitamin C and silver nitrate, and the regeneration efficiency was improved by promoting shoot formation. In addition, vitamin C and silver nitrate, which have the effect of preventing discoloration and promoting shoot formation, were individually and mixed with each other to reduce the incidence of mutagenesis in tissue culture of long - term Cymbidium plants
본 발명자는 PLB로부터 신초를 형성할 수 있는 재분화배지에 비타민 C와 질산은 첨가를 통하여 신초형성율을 증대시키고 갈변화를 감소하고 또한 비타민 C와 질산은 혼합첨가를 통한 변이체 발생빈도 감소에 대한 재분화 방법을 개발함으로써 본 발명을 완성하였다. The inventors of the present invention have found that regeneration method for reducing the frequency of mutation through addition of vitamin C and nitric acid by adding shoots of vitamin C and silver nitrate to the regeneration medium capable of forming shoots from PLB, Thereby completing the present invention.
본 발명은 심비디움의 재분화 체계 적용에 있어서 비타민 C와 질산은 첨가를 통하여 좀 더 효율적으로 심비디움 원괴체로부터 갈변화 감소 및 신초 형성율 증가 시키는 방법을 제공한다. 또한 갈변화 감소와 신초 형성을 증가시키는 비타민 C와 질산은의 최적의 농도 결정하는 것 외에도, 변이체 발생을 감소시키기 위하여 비타민 C와 질산은의 단독 및 혼합처리 중 효과가 우수한 처리구인 비타민 C와 질산은의 혼합처리를 통해 변이체 발생을 감소시키는 단계로 이루어진 것에 특징이 있다. 이를 통해 심비디움 우량묘의 기내 대량증식에 중요한 절편체인 PLB로부터 전반적인 신초 형성율 증가에 의한 재분화 효율 향상 뿐만 아니라 배양 중 발생하는 주요 문제점들인 갈변화 및 변이체 발생 현상 감소에 기여한다.
The present invention provides a method for reducing the change of color and increasing the shoot formation rate from the cymbidium raw body more efficiently through the addition of vitamin C and silver nitrate in the application of the regeneration system of cymbidium. In addition to determining the optimal concentration of vitamin C and silver nitrate to reduce galling change and shoot formation, it is also necessary to determine the optimal concentration of vitamin C and silver nitrate in the treatment of vitamin C and silver nitrate, And a step of reducing the occurrence of mutant through treatment. This result not only improves the regeneration efficiency by the increase of overall shoot formation rate from PLB, which is an important part for the large-scale propagation of Cymbidium-rich seedlings, but also contributes to the reduction of the occurrence of galley change and mutant phenomenon.
본 발명의 효과는 심비디움 원괴체 (PLB)로부터 비타민 C 및 질산은 처리를 통해 신초형성을 효율적으로 증가시키는 단계를 도입하고, 신초들이 대량으로 증식할 수 있는 체계를 구축하는 동시에 배양 과정 중에 흔히 발생하는 갈변화 및 변이 식물개체 발생을 감소시키기 위해 비타민 C 와 질산은을 배지에 첨가하여 과정에 의하여 보다 효율적인 재분화 체계를 확립하는 것이 가능할 것이다. 이러한 개선된 방법들은 다른 중요한 양란들인 카틀레야, 팔레놉시스 및 덴드로비움 등의 PLB로부터 재분화 향상 및 변이체 감소 연구에 적용되어 난과 식물들의 우량묘 기내 대량증식 체계확립에 기여할 수 있을 것이다.
The effect of the present invention is to introduce a step of efficiently increasing shoot formation through the treatment of vitamin C and nitric acid from the cymbidium source platelets (PLB), to establish a system capable of mass proliferation of shoots, It would be possible to establish a more efficient regeneration system by adding vitamin C and silver nitrate to the medium in order to reduce turf changes and mutant plant occurrence. These improved methods may contribute to the establishment of a mass proliferation system in plants that have been applied to the improvement of regeneration and mutant reduction studies from PLB such as cattleya, Palenopsis and dendrobium, which are other important angiosperms.
도 1은 심비디움의 분열조직에서 유도된 PLB(protocorm like-body)를 증식시키는 과정에서 고체배지와 액체배지에서의 생육 차이비교
도 2는 심비디움 분열조직으로부터 PLB (protocorm like-body)를 증식시켜 신초가 유도되는 과정
도 3은 PLB로부터 유도된 신초들이 발달하여 심비디움 기내 유묘로 생장 및 증식 중인 사진
도 4는 심비디움 PLB로부터 유래한 신초들이 기내 유묘로 생장 및 증식과정을 거친 후, 순화과정을 거쳐 화분에 이식되어 증식 중인 사진 FIG. 1 is a graph showing the difference in growth between solid medium and liquid medium during the proliferation of PLB (protocorm like-body) derived from the cleavage tissue of Cymbidium
FIG. 2 is a view showing a process in which shoot breeding is induced by proliferation of PLB (protocorm like-body)
FIG. 3 is a photograph showing PLB-derived shoots growing and propagating in Cymbidium in-flight seedlings
FIG. 4 is a photograph showing that the shoots derived from Cymbidium PLB have undergone growth and proliferation into seedlings in the cabin,
이하 비한정적인 실시예를 통항여 본 발명을 더욱 상세하게 설명한다. 단 하기 실시예는 본 발명을 예시하기 위한 의도로 기재된 것으로서 본 발명의 범위는 하기 실시예에 의하여 제한되는 것으로 해석되지 아니한다.
The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the invention and the scope of the invention is not to be construed as being limited by the following examples.
실시예 1: PLB로부터 신초형성 실험을 위한 원괴체 증식Example 1: Platelet proliferation for shoot formation test from PLB
본 발명에 사용된 심비디움 식물체는 농촌진흥청 산하 국립원예특작과학원에서 분양 받은 융플라워X마사코 계통을 사용하였다. 심비디움 원괴체 (protocorm-like body;PLB)를 증식하기 위해 줄기 없이 PLB만 일정크기(가로세로 0.5 cm)로 자른 후에 PLB 증식배지 [HCa배지: Hyponex 3 g/l, trypton 1.5 g/l, CaNO3 0.5 g/l, sucrose 20 g/l, 활성탄 1 g/l, Agar는 7.5 g/l]인 HCa배지에 이식하였다. 모든 배지의 pH는 5.2로 맞추었다. 상기 과정을 거쳐 증식된 PLB를 고체배지와 액체배지 중 어느 배지가 증식과 재분화에 더 효율적인지 알아보기 위해 비교실험을 수행하였다. 고체배지는 상기 증식배지와 동일한 배지이고 액체배지는 상기 고체배지에서 agar만 제외하고 제조한 배지이다. 또한 계대배양 주기의 차이를 알아보기 위하여 액체배지는 1주, 2주 및 4주간격 그리고 고체배지는 2주와 4주 간격을 계대배양 하여 증식율을 비교하였다. 표 1은 액체배지와 고체배지 이 두 가지 서로 다른 형태의 배지가 PLB 증식에 미치는 영향에 대한 결과를 나타낸 것인데, 2주 간격으로 계대배양한 처리구가 가장 우수한 생육을 보여 주었고, 2주 간격으로 계대배양 시, 두 종류의 배지를 비교한 결과, PLB 증식은 액체배지에서 고체배지보다 생체중이 1.96배 더 증가했음을 보여주었고, 건물중에 있어서도 2.14배 더 증가하여 액체배지에서 PLB 증식이 생체중과 건물중 조사 결과, 더 우수함을 보여 주었다. 그러나, 증식효율만 고려하면 액체배지가 고체배지보다 거의 2배 가까이 우수하나, 증식 후 재분화 하는 과정에서 더 우수한 지는 추가연구가 필요하다.
The Cymbidium plants used in the present invention were purchased from the National Institute of Horticultural Science under the Rural Development Administration. PLB growth medium [HCa medium: Hyponex 3 g / l, trypton 1.5 g / l, CaNO (0.5 g / l)] was prepared by cutting PLB without any stem to a certain size 3, 0.5 g / l, sucrose 20 g / l, activated carbon 1 g / l, and Agar 7.5 g / l). The pH of all media was adjusted to 5.2. A comparative experiment was conducted to determine whether PLB, which was propagated through the above process, was more efficient in proliferation and regeneration of the solid medium and the liquid medium. The solid medium is the same medium as the above-described growth medium, and the liquid medium is a medium prepared by excluding agar alone from the solid medium. In order to investigate the difference of subculture period, the growth rate of liquid medium was compared with that of subculture at 1, 2, and 4 weeks intervals and solid medium at 2 and 4 weeks intervals. Table 1 shows the effect of two different types of medium on the PLB proliferation of the liquid medium and the solid medium. The subculture treated with subculture at intervals of 2 weeks showed the best growth, The PLB proliferation was 1.96 times higher in the liquid medium than in the solid medium and 2.14 times higher in the PLB culture than in the solid medium. The results showed that it was better. However, considering the proliferation efficiency, the liquid culture medium is nearly twice as much as the solid culture medium, but further studies are needed to see whether it is better in the process of regeneration after proliferation.
표 1은 PLB 증식에 고체배지와 액체배지 효율비교
Table 1 shows the comparison of solid medium and liquid medium efficiency for PLB growth
실시예 2: PLB로부터 신초형성 촉진 및 갈변화 감소에 비타민 C와 질산은 처리 Example 2: Vitamin C and nitric acid treatment for promotion of shoot formation and reduction of browning from PLB
PLB로부터 식물체로 재분화하기 위해 심비디움 증식배지인 HCa 기본배지로 옮겨 소식물체의 재분화를 유도하였다. 이 기본배지에 비타민 C은 0, 25, 50, 75, 100, 150 , 200 mM 농도로 첨가하였고, 질산은은 0, 2.5, 5, 10, 15, 20, 25 mM 농도로 첨가하여 갈변화 감소와 신초형성 촉진 효과를 비교하였다. 표 2는 이러한 비타민 C와 질산은 처리가 갈변화 감소 및 신초 형성에 미치는 영향에 대한 결과를 나타낸 것이다. 배지에 비타민 C 150 mM 처리 시 무처리구와 비교하여 갈변화는 절반이하로 줄어 들었는데, 질산은의 경우 20 mM 첨가 시 무처리구와 비교하여 1/3 이하로 감소하여 질산은 20 mM 처리가 가장 효과적임을 보여 주었다.
In order to regenerate from PLB to plant, it was transferred to HCa basic medium, which is a cymbidium growth medium, to induce regeneration of microbial bodies. Vitamin C was added to the basal media at 0, 25, 50, 75, 100, 150 and 200 mM concentrations and silver nitrate was added at 0, 2.5, 5, 10, 15, The effects of promoting shoot formation were compared. Table 2 shows the results of the effects of these vitamin C and nitrate silver treatments on the reduction of browning and shoot formation. In the case of 150 mM of vitamin C, the change of browning was reduced to less than half of that of control. The addition of 20 mM of silver nitrate decreased to less than 1/3 of that of untreated one and 20 mM of nitrate was most effective.
표 2는 질산은 및 비타민 C 처리가 갈변화 감소 및 신초형성에 미치는 영향
Table 2 shows the effect of silver nitrate and vitamin C treatment on the decrease of browning and shoot formation
실시예 3: 비타민 C와 질산은 처리를 통한 심비디움 기내 변이체 식물 감소Example 3: Reduction of Cymbidium In Vitro Mutants Plant by Treatment with Vitamin C and Silver Acid
심비디움 대량 증식 과정 체계를 확립하는 부분에서 갈변화 감소 및 신초형성의 증가도 중요한 요인들이지만 증식된 심비디움 기내식물체들에 있어서 변이체 발생을 감소시켜 증식되는 것도 중요한 부분이다. 따라서 갈변화 현상의 감소에도 효과가 있고 신초 형성 증대를 통한 재분화 효율 향상에 효과를 보여 주었던 비타민 C와 질산은 처리가 이러한 변이체 감소에 미치는 영향을 알아보고자 실험을 수행하였다. 이를 위하여 신초 재분화 목적으로 사용된 PLB 증식배지에 갈변화 감소와 신초 형성에 가장 우수한 효과를 보여 주었던 비타민 C 150 mM, 질산은 20 mM, 두 가지 항산화제의 혼합처리 그리고 무처리구 이렇게 처리하여 비교한 결과를 표 3에 나타낸 것이다. 비타민 C와 질산은 단독처리구는 무처리구와 비교해서 크게 개선된 효과를 보이지 않았으나, 비타민 C와 질산은을 혼합한 처리구는 무처리구와 비교해서 1/2 이하의 변이체 감소효과를 보여 주었다. It is also important to reduce the number of varieties and increase the number of shoots in the process of establishing the Cymbidium mass proliferation system, but it is also important to reduce the occurrence of mutants in the propagated Cymbidium in-flight objects. Therefore, the effect of vitamin C and silver nitrate treatment on the reduction of mutant was investigated. For this purpose, PLB growth medium used for shoot regeneration was supplemented with 150 mM vitamin C, 20 mM silver nitrate, which had the best effect on reducing the browning change and shoot formation, As shown in Table 3. Vitamin C and nitric acid alone treatments did not show any significant improvement compared to untreated control. However, treatment with vitamin C and silver nitrate showed less than half the mutagen reduction compared to untreated control.
표 3은 비타민 C, 질산은 및 비타민C + 질산은 혼합처리가 심비디움 기내변이체 Table 3 shows that the mixing of vitamin C, silver nitrate and vitamin C +
발생빈도에 미치는 영향
Effect on frequency of occurrence
실시예 4: PLB로부터 형성된 신초로부터 식물체 재분화 과정 Example 4: Plant regeneration from shoots formed from PLB
본 발명에 재분화된 소식물체는 같은 HCa 배지로 한 달에 한 번씩 옮겨주고, 25℃, 광주기 16/8(명/암)시간 조건을 통하여 신장과 뿌리발달을 통한 정상적인 식물체 재분화가 이루어졌다. 고체 배지 상에서 신초 형성과 발근이 완료된 식물체는 남아 있는 agar 조각을 제거한 다음 원예용 수태를 이용하여 화분으로 이식하였다. 본 발명에서 위의 모든 배양은 25℃, 16시간 조건의 광주기를 주었다. In the present invention, the repopulated microorganisms were transferred to the same HCa medium once a month and normal plant regeneration was carried out through the kidney and root development at 25 ° C and light period of 16/8 (person / cancer) time. After planting and rooting on the solid medium, the remaining agar pieces were removed and then planted in pots using horticultural fertilization. In the present invention, all of the above cultures were incubated at 25 DEG C for 16 hours.
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| CN107750949A (en) * | 2015-07-22 | 2018-03-06 | 浙江传化生物技术有限公司 | The method that hybrid cymbidium seedling is efficiently bred using stem-tip tissue |
| CN108849504A (en) * | 2018-06-20 | 2018-11-23 | 福建农林大学 | A method of hybridization sword-leaved cymbidium rhizomes floral bud induction is at colored |
| CN109006472A (en) * | 2018-06-12 | 2018-12-18 | 杭州电子科技大学 | The method for carrying out ornamental plant tissue-culturing rapid propagation using certain concentration carbon nanotube |
| CN112640784A (en) * | 2021-01-07 | 2021-04-13 | 永州市农业科学研究所 | Artemisia apiacea one-time seedling tissue culture method with low mutation rate |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107750949A (en) * | 2015-07-22 | 2018-03-06 | 浙江传化生物技术有限公司 | The method that hybrid cymbidium seedling is efficiently bred using stem-tip tissue |
| CN109006472A (en) * | 2018-06-12 | 2018-12-18 | 杭州电子科技大学 | The method for carrying out ornamental plant tissue-culturing rapid propagation using certain concentration carbon nanotube |
| CN109006472B (en) * | 2018-06-12 | 2020-06-16 | 杭州电子科技大学 | Method for tissue culture and rapid propagation of ornamental plants by using carbon nano tubes with specific concentrations |
| CN108849504A (en) * | 2018-06-20 | 2018-11-23 | 福建农林大学 | A method of hybridization sword-leaved cymbidium rhizomes floral bud induction is at colored |
| CN112640784A (en) * | 2021-01-07 | 2021-04-13 | 永州市农业科学研究所 | Artemisia apiacea one-time seedling tissue culture method with low mutation rate |
| CN112640784B (en) * | 2021-01-07 | 2022-06-03 | 永州市农业科学研究所 | Artemisia apiacea one-time seedling tissue culture method with low mutation rate |
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