농진청에서 수행하고 있는 아프리카 국제협력프로그램인KAFACI 사업의 일환으로 우간다 등 8개국에 ‘통일형 다수성벼 품종개발’ 사업을 추진하고 있다. 이를 위해 아프리카에서정상적인 생육과 수량을 나타내는 다수성 통일형 품종인 밀양23호와 아프리카의 재래종인 O. glaberrima를 이용하여 밀양23호의 유전적배경을 보유한 근동질 계통인 BC4F1을 육성하고, 약배양을 통해 유전적 고정계통을 육성하였다. 이 중 50개계통을 우간다에서 생물검정을 실시한 결과, 아프리카에서 문제시되는 Yellow Mottle Virus (RYMV), Bacterial LeafStreak(BLS), 흰잎마름병 및 도열병에 복합저항성인 계통으로판명되었다. 특히 RYMV에 대한 저항성은 저항성원이 결여되어 있는 병으로 본 연구를 통해 육성한 계통들은 향후 아프리카에 적응하는 내병성 다수성 품종개발에 유용한 재료를 활용 될 것으로 생각된다.

벼의 약 및 현미 배양효율과 관련된 DNA marker를 이용하여 인디카형 벼 품종인 'IR 36'의 조직배양 효율을 개선하기 위하여 실험한 결과를 요약하면 다음과 같다. 벼품종 간에 약 및 현미배양 효율을 비교한 결과 자포니카 > 통일형 > 인디카 형의 순으로 나타났다. 그러나 MGRI집단의 약배양에서 식물체분화율이 높은 계통으로 선발된 'MGRI 079'와 'MGRI 036'의 약배양 효율은 각각 19.8%, 19.9%로 가장 높게 나타났다. 'MGRI 079'에 'IR 36'이 여교배되어 양성된 90 계통에 대한 marker검정을 실시하여 positive band를 나타내는 34계통을 선발할 수 있었다. 선발된 34계통 중 10 계통의 약배양에서 캘러스 형성률은 'IR 36' 보다 현저히 높았다. 선발된 10 계통의 현미배양에서도 캘러스형성 능력과 식물체재분화율이 'IR 36' 보다 높게 나타났다. 계통 중에서 식물체분화능력이 높은 계통으로 선발된 -28은 간장이 'IR 36'보다 큰 편이었으나 출수기와 미립특성은 'IR 36'과 비슷하였다.

캘러스, 반수체, picloram, 전처리양식을 규명할 목적으로 배양효율에 미치는 화분발육 단계와 온도전처리가 미치는 영향에 대한 실험결과는 다음과 같다. ‘Dreamland’의 감수분열중인 약을 현 미경적방법으로 검경하여 본 결과 화뢰의 길이가 23.0-24.9mm는 tetrad 단계이었고 25.0-26.9mm는 uninucleate 단계, 27.0-28.9mm에서는 binucleate 단계 임을 확인할 수 있었으며, 저온 및 고온 온도처리 결 과는 전체적으로 캘러스 및 배 발생율은 저조하였고 특 이한 점은 발견할 수 없었다. Microgametogenesis 단 계별로 약배양 효율을 조사한 결과, late uninucleateearly binucleate 단계인 23.0-28.0mm에서 17.8% callus가 유도되었고, 식물체 재분화율도 6.7%로 나타 나 적정 배양 stage임을 확인할 수 있었다. 유기된 캘 루스를 picloram과 zeatin을 혼용한 MS 배지에 이식 하여 25oC에서 배양하여 식물체를 얻었다.

이탈리안 라이그라스 (Lolium multinorum Lam.)와 톨페스큐 (Festuca arundinacea Schreb.)의 속간 교잡종 3계통에 대한 약 배양을 실시하여 약 유래의 식물체를 생산하였다. MS 기본배지에 sucrose 30 g/, NAA 2 mg/, kinetin 1 mg/을 첨가한 캘러스 유도배지에서 배양 20일만에 약 유래의 캘러스를 유도하였다. 캘러스 유도율은 평균 11.6%였으며, 캘러스의 크기는 평균 9.1 mg/callu

이탈리안 라이그라스(Lolium multiflorum Lam.) 계통 A의 약(葯) 배양을 실시하여 약(葯) 유래의 식물체를 생산하였다. MS 기본배지에 sucrose 30 g/, NAA 2 30 mg/, kinetin 1 30 mg/을 첨가한 캘러스 유도배지에서 배양 20일만에 약(葯) 유래의 캘러스를 유도하였다. 캘러스 유도율은 9.2%였으며, 캘러스의 평균 크기는 8.6mg/callus/anther 였다. MS 기본배지에 sucrose 30 g/,

본 연구는 Petunia hybrida hybrid를 약배양(葯培養)하여 반수체(半數體)를 얻을 목적으로 약(葯)의 치상시기(置床時期)와 배지(培地)의 종류(種類), 생장조절물질(生長調節物質)의 종류와 농도, 약(葯)의 전처리(前處理)와 품종(品種)에 따른 캘러스 형성률을 조사하였던 바 다음과 같은 결과를 얻었다. 약배양하기 전 0.8-1.2cm의 길이의 화뢰(花雷)가 달린 모본(母本)의 줄기를 에서 15일간 저온처리(低溫處理)하였다. 약을 채취하여 1/2 MS배지에 NAA 5.0mg/, BAP 0.5mg/를 첨가한 배지(sucrose 30g/, 2g/, pH 5.8)에서 배양 4주후 연녹색의 캘러스가 형성되었으며, 이들 캘러스로부터 배양 3주후 2ip 2.0mg/가 함유된 MS배지에서 식물체가 분화되었다.

Background : Korean ginseng require 3 - 4 years to produce mature seeds from their mother plants. Therfore, it takes over 20 years to genetic fixation by artificial crossing of 8 generation. Anther culture is a useful method for obtaining homozygotes in only one generation. However, there is not much research on ginseng yet. In this study, we investigated the callus induction of anther depending on the type and concentration of the plant growth regulators in Panax ginseng C. A. Meyer. Methods and Results : Flower buds of P. ginseng were cold pretreated at 2 days before the anthers were plated on the induction medium. The flower buds were immersed in 70% ethanol for 30 sec min, washed two times with sterile distilled water, surface-sterilized in 2% sodium hypochlorite solution for 20 min, then rinsed five times with sterile distilled water. The anthers were placed on Petri dishes containting fifteen different concentrations and combinations of 2,4-D, NAA, BAP and KT. Callus induction was significantly influenced by the type and combination of plant growth regulators. The highest callus induction rate was observed in GR5 medium at 79.2%. The 2,4-D mediums had significantly higher callus induction than the NAA medium, and 2,4-D 1 ㎎/ℓ have a higher callus induction rate than the other concentrations. The increase of callus induction rate was not observed by the addition of cytokinin, but the callus induction rate was gradually decreased as the BAP concentration was increased. There was no difference in callus induction rate between BAP and KT. Conclusion : The important factor for inducing callus of ginseng anther was the addition of 2,4-D, and no effect of cytokinin addition could be found.

Anthocyanins are the major pigments contributing to flower coloration. A 1584 bp 5' upstream sequence of ALCHS2 gene was isolated from Acapulco lily (Lilium Oriental hybrid cv. Acapulco). Computer-based analyses (GeneScan, AtPAN) predicted a CAATBOX1 and putative transcription factor-binding sites, including tissue-specific elements. When gALCHS7 promoter–gus fusion was introduced to petunia ('Dream Red'), all ten putative transgenic plants showed localized GUS activity in the anther, but five of them also showed weak GUS activity in the ovule. No distinctive signal in the leaf and petal was detected in the same stage. To clearly determine the operation of the promotor region, anther and ovule tissues of transgenic line 6 were fixed in paraffin for dark-field analysis. At 1 cm length of floral bud, a GUS signal was not observed in the anther, but weak expression was observed in the ovule. Before anthesis, GUS protein was highly expressed in the pollen, endothecium, and epidermis. Fluorometric GUS assays of individual organs taken from four transgenic plants demonstrated that all lines showed high GUS activity in the anther compared to 35S CaMV promoter (pBI1 121), except line 34. Using the truncated promoters by cis-acting elements, we found that minimal region (gALCHS7-7, 270 bp) displayed GUS expression only in the anther, though at weaker activity than in the original promoter.

Early maturing glutinous rice lines with giant embryo were developed using anther culture. Deuraechan, mid-late maturing high-yielding japonica rice variety with resistance against rice stipe virus (RSV), bacterial blight (BB), and lodging, and Chenghyangna ge, early maturing glutinous rice germplasm with giant embryo were used the parents. F2 seeds from the cross between Deuraechan and Chenghyangna ge with glutinous endosperm and giant embryo were selected and propagated to F2 population. In F2 population, anther culture was conducted using the panicles from the early maturing plants. All doubled haploid (DH) lines showed early maturing, glutinous endosperm, and giant embryo phenotype. Through marker-assisted selections to Stvb-i and Xa3, 17 DH lines carrying both resistance genes were selected. Among 17 DH lines, six lines with more embryo size and better agronomic traits were selected and analyzed their characteristics. These lines were early maturing glutinous rice with giant embryo and showed enhanced yield, resistance against RSV and BB, and lodging, compared to previously developed giant embryo rice varieties. But they were vulnerable to preharvest sprouting which is important trait in early maturing rice. According to the texture and rapid viscosity analysis, DH lines were considered to have appropriate properties of cooked brown rice. They showed less hardness, gummniess, chewiness, and setback. Developed DH lines could be useful materials for diversification of cropping system and enhancing the brown rice consumption but the breeding efforts to improve the vulnerability against preharvest sprouting is required to apply for practical variety.

Anther culture is useful and significant tool for producing haploid or doubled haploid (DH) plants in crop breeding system. Androgenesis is the way of inducing haploid and DH plants from anther (immature pollen) or microspore culture. In vitro androgenesis is efficient technique for introducing complete homozygous lines in one generation, thus less time and expense could be necessary than conventional plant breeding. In maize, anther culture is important system for shortening the breeding cycle and enhancing selection efficiency. Anther culture technique is also applicable to various researches such as molecular genetics, genetic engineering, genomics, and plant biotechnology. We review the past and present studies on anther culture and provide useful information for future researches on androgenesis in maize. The combination of androgenesis with other techniques such as molecular breeding and biotechnology is producing a variety of variety of maize species. In addition, we suggest strategy to develop androgenesis technique adapted to Korean research environment.

In rice, the stage of the meiosis in the pollen is sensitive stage resulted in the pollen sterility to reduce yield. Dianxi4 is a cold tolerant line. To monitoring the proteome expression patterns in the pollen of Dianxi4 under the cold stress, shotgun proteomic analysis was conducted to the anther of Dianxi4. The rice plant was grown in the peedy rice field then in the 10 DBH(days before heading), one individual rice plant was moved in the growth chamber under the condition of12℃/RH70%(12h day/12h night). Also the plant used as control was moved in the growth chamber unde the condition of 28℃/RH70%(12h day/12h night). after 4 days treatment, the plant were moved in a greenhouse. The treated rice anther were collected in the one day before heading. From the shotgun proteomic analysis, total of 3,855 non-redundant proteins were identified. Among them, 2,360 proteins were reproducibly identified through the treatment and replications. By the T-test, 1,181 differentially expressed proteins were detected. Through the GO analysis, proteins related in gene expression, cellular process, cellular biosynthetic process were enriched.

당근의 약배양을 통한 효율적 식물체 재생 시스템을 확립하고자 실험을 수행하였다. 당근 유전형 ‘S&P2342’의 약을 2,4-D와 NAA를 각각 0.1 mg/L 또는 1.0 mg/L 단독 또는 조합 처리한 배지에 치상하여 암조건에서 18주간 배양하였을 때, 2,4-D 1.0 mg/L + NAA 0.1 mg/L 조합처리에서 캘러스 형성율 22.0%, 배형성율 2.0%로 반응이 가장 좋았다. 약으로부터 직접적으로 유도된 1차배를 분리하여 명조건에서 2주간 배양 후 재생배지로 옮겨 배양하였을 때 배양 4주 후부터 발아하다가 생장이 멈추어진 1차배로부터 다수의 2차배가 형성되기 시작하였다. 배양 8주 후 62.5%가 식물체로 전환되었으며, 12주 후 본엽이 전개된 소식물체로 발달하였다. 또한 약으로부터 유도된 캘러스를 분리하여 재생배지에서 배양하였는데, 8주 후 93.9%의 캘러스로부터 1차배가 형성되었으며, 38.8%에서 1차배로부터 2차배 형성을 거쳐 식물체로 전환되었다. 약배양 유래 소식물체(본엽 2매 이상, 초장 2-4 cm)의 건전한 기내 생장을 유도하기 위해 실시한 광도(10, 30, 100, 150 μmol·m-2·s-1) 처리에서 100 μmol·m-2·s-1이 소식물체의 생장에 가장 효과적이었다. 소식물체의 효율적인 기외 활착을 위하여 본엽 4매 이상, 초장 5 cm 이상의 소식물체를 플러그 트레이에 이식 후 플라스틱 재배용기 안에 넣은 다음 온도 (15, 20, 25℃)와 광도(μmol·m-2·s-1) 처리를 각각 2주간에 걸쳐 실시하였는데, 온도 처리에서는 15℃가 기외 활착에 가장 적합하였으며, 광도 처리에서는 100 μmol·m-2·s-1 처리구만 기외 활착이 원활하였다. 결론적으로 당근의 약배양으로부터 직접 배형성 또는 캘러스 단계를 거치는 간접 배형성이 이루어졌으며, 1차배로부터 2차배가 형성된 후 발아를 거쳐 소식물체가 재생되었으며 이를 성공적으로 활착시켜 정상적인 식물체를 얻을 수 있었다. 본 연구를 통하여 당근에서의 약배양으로부터 직접 및 간접 배형성을 통한 식물체 재생 시스템을 구축하였으며, 향후 당근의 F1 잡종 생산을 위한 순수 동형접 합성 유지계통 및 화분친 모본계통의 획득에 활용할 수 있을 것으로 생각된다.

Rice is one of the most important cereal crops in the world and a model plant for functional genomics of monocotyledon. Recently, rice crop loss is estimated to be approximately 30% of the total yield due to herbivorous pests, mainly insects. Cry1Ac toxin is a protein produced by the bacterium Bacillus thuringiensis and has insecticidal properties. CryBP1 toxin also is an insecticidal protein produced by the bacterium Bacillus popilliae. These two toxic genes derived bacteria, which were inserted into a binary vector, have been introduced into rice plants by Agrobacterium tumefaciens mediated transformation in order to enhance resistance to insects. Here, we utilized anthers to regenerate transgenic rice plants when it has been plated on the callus induction media as a callus-inducing material. Anther culture has a benefit in terms of being apt to produce doubled haploids in short term in plants breeding compared to seed culture. However, anther culture method in generating transgenic rice still has low productivity of plant regeneration in some genotypes of Japonica rice. Therefore, we examined the efficiency of callus induction and transformation with three different cultivars of Japonica rice, Haiami, Ungwang and Dongjin. In this study, our results showed that Haiami is the best genotype among three cultivars of Japonica rice as callus inducing material in anther culture to produce transgenic rice plants conferring resistance to insects.

본 연구는 벼멸구, 벼흰잎마름병, 벼줄무늬잎마름병에 저항성인 자포니카 복합내병충성 품종을 조기에 확보하고자 약배양을 수행하여 목표 저항성 유전자 조합의 우량 고정계통을 개발하고 육성 과정 중에 발생할 수 있는 문제점을 파악하여 병해충 저항성 육종사업에 반영하고자 수행하였다. 벼흰잎마름병과 벼줄무늬잎마름병에 저항성인 우량 계통 HR26234- 12-1-1과 벼멸구, 벼흰잎마름병, 벼줄무늬잎마름병에 저항성인 SR30071-3-7-23-6-2-1-1을 인공 교배한 F1을 약배양하여 213개 고정계통을 육성하였다. HR26234는 Xa3+xa5, Stvb-i, SR30071은 Bph18, Xa4, Stvb-i를 가지고 있는 것으로 확인되었다. 이들 유래 약배양 계통들은 모두 Stvb-i를 가지고 있었고, Bph18+Xa3, Bph18+Xa4, Bph18+Xa3+xa5, Bph18+Xa4+xa5, bph18+Xa3, bph18+Xa4, bph18+Xa3+ xa5과 bph18+Xa4+xa5의 조합이 확인되었다. 약배양 집단에서 xa5+Bph18(또는 bph18)+Stvb-i 조합이 발생하지 않았고 Bph18 보유 계통이 bph18 보다 적게 발생하는 등 segregation distortion이 발생하였다. Bhp18을 보유하며 벼멸구에 저항성인 계통들이 감수성인 계통들에 비해 간장이 컸다. 선발된 Bph18+Xa4+xa5+Stvb-i 조합의 계통은 단간이면서 벼멸구, 벼흰잎마름병, 벼줄무늬잎마름병에 저항성을 나타내었으나, 낮은 수량성과 일부 불임의 발생, 단간임에도 도복에 안정적이지 못한 특성을 나타냈다. 병해충에 대한 저항성 육종사업에서 약배양을 활용하여 조기에 육종 목표를 달성하고자 할 경우에 segregation distortion이 발생하여 편의 된 변이가 발생할 수 있고, 저항성 유전자 도입 시 linkage drag에 의해 예기치 못한 열악형질 특성이 나타날 수 있음을 고려하여 신중하게 목표에 접근하여야 할 것으로 생각한다.

A male gametophyte, or pollen develops in the anther, and its development plays an important male reproductive process in flowering plants. A properly designed transgene construct can help to tailor transgene expression in plants by altering the expression strength, timing, and location. In this process, the promoter plays a pivotal role in controlling transgene expression. In this research, the promoter regions of rice anther/pollen-specific genes, named as OsMSP1 to OsMSP11,were selected from the microarray data sets covering 4 developmental stage of male gametophyte and then used for the construction of vector by Gateway cloning method and transformed into rice and Arabidopsis. All 11 promoters in rice and 9 in Arabidopsis were displayed as anther/pollen-specific/preferential genes by GUS assay and RT-PCR analysis. Three out of 11 promoters showed consistent results with published data. In this study, we demonstrated on eight new anther/pollen-specific or -preferential promoters (OsMSP1, OsMSP2, OsMSP3, OsMSP4, OsMSP5, OsMSP6, OsMSP8, and OsMSP9, which have not been reported before. Although the expression pattern of different genes active in pollen grains is diverse and complex, these experimental results would be helpful to understand the molecular mechanism of regulatory elements in rice microspore/pollen-specific genes.

This study was set up to get plants from anther culture of Chrysanthemum (Dendranthema grandiflorum) gardenmum cultivar “Yes Morning’ and potmum cultivar “Peace Pink” for breeding program. The induction of callus was quick and high on MS basal medium supplemented with 1.0 mg/L of 2,4-D + 2.0 mg/L of 6-BA + 4% W/V sucrose. Induction potential was slightly increased by addition of 250 mg/L Casein hydrolysate to the induction medium. Calluses were allowed to differentiate on MS basal medium + 2.0 mg/L of BA + 0.1 mg/L of NAA + 3%W/V sucrose. The rate of callus formation differed little between the cultivars. A pretreatment of anthers at 4℃ for 48h enhanced both the induction and differentiation ratio. Multiple shoots were initiated from most of the calluses and were shifted to MS basal medium + 0.1 mg/L of NAA + 3%W/V sucrose for rooting. Regenerated plantlets were acclimatized and transferred to the soil. Some of the regenerated plants showed slow growth with little morphological difference.

The research concerned of the regeneration of plants from embryos obtained from anther cultures of ginseng (Panax ginseng C. A. Meyer). The aim was to determine the influence of the regeneration medium on the efficiency of the regeneration process. We conducted to determine the optimum conditions such as cold pretreatment, plant growth regulators and carbon sources on anther culture of P. ginseng. Highest callus formation rate was obtained when flower buds pretreated at 4℃ for 1 day. Among the treated growth regulators with various degrees of concentration in Murashige and Skoog's (MS) medium, 4.53 μm of 2.4-dichlorophenoxyacetic acid and 4.44 μm of 6-benzylaminopurine gives the most responsive callus with the frequency of 73.89% and 129.53 g of fresh weight. When we used 3-9% of sucrose and maltose among the different kinds and various concentrations of carbohydrates, callus was formed highest 67.29% in the medium with 3% of sucrose. Shoots induced from callus supplemented with 28.9 μm of gibberellic acid and rooted in Gamborg's B5 medium supplemented with 14.7 μm of indole-3-butyric acid.