작약(芍藥)의 약(葯)으로부터 분리된 화분소포자(花粉小胞子)를 생장조절제가 첨가되지 않은 MS 배지에 배양하였을때 직접 배(胚)발생 과정을 거쳐서 소포자 유래의 배(胚)가 형성되었고, 1mg/l의 PAA가 첨가된 배지에서는 화분소포자 유래의 캘러스가 형성 되었으며 이들 캘러스를 생장조절제를 함유하지 않은 배지에 이식하였을 때 배양 7주 후에 배(胚)가 형성되었다.

We recently reported rice promoters that are active in late stages of pollen development. However, rice promoters that allow manipulation of gene expression at earlier stages of pollen development are still very limited to date. In this study, we have chosen 10 putative microspore promoters, OsMSP1 through OsMSP10, based on publicly available transcriptomic datasets in rice (Oryza sativa L.). Sequence analysis of these promoter regions revealed some cis regulatory elements involved in pollen-specific expression. We also examined promoter activities using the promoter-GUS reporter constructs in both transgenic rice and Arabidopsis. In rice, all of the 10 promoters directed GUS signals from the microspore stage throughout the all stages of pollen development. In addition, while GUS signals from 4 promoters, OsMSP2, OsMSP7, OsMSP9 and OsMSP10, seem to be expressed preferentially during pollen development, those from other six promoters were observed in vegetative tissues such as leaves, stems, and roots of seedlings. Similarly, in Arabidopsis, all of the 10 promoters directed GUS signals during pollen development. In detail, 8 promoters, OsMSP1 ~ OsMSP8 directed GUS signals from the microspore stage, whereas 2 promoters, OsMSP9 and OsMSP10, exhibited GUS signals from tricellular stage. Furthermore, seven promoters, except for OsMSP1, OsMSP2 and OsMSP10, showed GUS signals in shoot apical region or root tissues of seedlings. Furthermore, we verified microspore activity of four promoters, OsMSP1, OsMSP2, OsMSP3 and OsMSP6, by complementation analysis of the sidecar pollen (scp) mutant which displays microspore-specific defects. Currently, further analyses are underway for GUS expression of T2 generation in transgenic rice and scp complementation with remaining promoters.

Based on the results of microarray analysis we selected ten candidate genes that express in pollen at the early pollen developmental stage. By PCR amplification, the promoter region of these genes were amplified from rice genomic DNA (Nipponbare) and cloned into the destination pKGWFS7 vector via an entry vector, pDONR201. The characteristic of promoters were evaluated in Arabidopsis thaliana (Col-0) through GUS expression analysis. Fifty T2 plants respectively from each promoter were tested. Whole inflorescence of individual plant was stained with 1mM X-Gluc solution to observe tissue-specific GUS expression patterns. The results showed that all 10 promoters activated in pollen tissues. Among them six promoters expressed at the early developmental stage (unicellular) of pollen and the others expressed at both early (unicellular) and late pollen developmental stage (mature pollen). The results indicated that these promoters would be potential applicable for the studies of pollen function. Currently, we are performing these promoters analysis in rice transgenic plants as well as molecular characterization.

Glucosinolates (GLS) are secondary metabolites commonly occurring in Brassica crops and more than 130 different GLS have been reported in diverse plants. Recent studies have indicated that isothiocyanate (ITC) derived from GLS by hydrolysis had a potential for anticancer activity against several rumor cells on human. In addition, it was found that glucoraphenin (GRE) and glucoraphasatin (GRH) were abundant and differently regulated in radish plant, depending upon organs and developmental stages. Microspores isolated from flower buds of radish were cultured in vitro to obtain doubled haploid (DH; but homozygous) lines in a short time period. The present study was conducted to determine the concentration of GRE and GRH, an immediate precursor of ITC from DH lines of radish plant. Total 41 DH lines were selected based on flow cytometry analysis. The seeds, obtained by bud pollination from the DH lines, were planted and 3-weeks-old young seedlings were used for the major aliphatic GLS analysis. Amounts of GRH were highly variable from the DH lines ranging from 2.3 to 31.5 mg·g-1 dry weight (DW). The donor plant (DP) contained 18.4 mg·g-1 DW. It was noticed that there were 6-fold differences in the amounts of GRE between the highest and lowest DH lines. Among 41 lines tested, 14 DH lines of radish plant were significantly reduced in the amount of sum of GRH and GRE compared those of the donor plant (P<0.05), whereas only three lines increased. The results obtained in the present study will lend to select genotypes with low and high GLS contents of radish plant. In addition, those DH lines will aid to elucidate a biosynthetic pathway of the aliphatic GLS in radish plant, which remain for the most part unsolved.

Investigation of flowing time, flower structure, microspore density, microspore vitality and microspore-derived embryo (MDE) formation rate according to the light quality treatment on broccoli donor plant was accomplished. The material was 08-8-3 line yielding high MDE production rate having 4.0 ± 0.5 mm flower bud length. The donor plant was cultivated with light quality treatment of red LED light, red+blue+white LED light and fluorescent light. The light intensity was 50 μ molm-2s-1 and photoperiod was 16/8 hours (light/dark). The flowering time was fastest at red LED light treatment compared to the other light treatment condition. 100.0, 36.4 and 18.2% of flower bud with longer stigma length than floral leaf which reported high MDE production rate were found under red LED light, Red+Blue+White LED light and fluorescent lights respectively. The microspore density and MDE production rate per single flower bud was highest at Red LED light. Suitable flower bud and high MDE production rate could be achieved in a short period if using LED light to broccoli donor plant cultivation. The above result is thought to be very useful for the development of a new cultivar of broccoli and other many crops including Brassica using haploid breeding technology. This journal was supported by the National Research Foundation

Heat shock pretreatment, dark culture period and washing medium could have marked effects on microspore embryogenesis. A heat shock pretreatment of microspores at 32.5°C for 48 hours gave high production rate of microspore-derived embryo (MDE) when compare to shorter and longer period. The yield of MDE increased significantly when microspore cultured for 15 days at 25℃ in dark condition followed by heat shock pretreatment. MDE were browned and lost vitality when dark treatment period extended longer than 15 days. This is caused by an insufficient oxygen and light for growing embryo which already formed during dark treatment period. The vitality of a microspore isolated from flower bud stored at 4℃ become decreased at the very first day and the vitality of microspore stored at 4℃ in the form of flower bud itself become decreased from the 5th day after storage. This shows the possibility of getting a certain period of storage for a suitable flower bud in MDE formation. The yield of MDE was most effective when isolated microspore was had with MS medium compared to B-5 and NLN medium and also showed most effective result with sucrose 130 g􀂂L-1 in additional sucrose concentration. The above result is thought to be very useful for the development of a new cultivar of radish and other many crops including Brassica using haploid breeding technology.

브로콜리 74 계통 화기 구조를 통한 소포자 배 발생을 살펴본 결과 18개 계통에서 소포자 유래 배 발생이 확인되었다. 특히 08-8-8 계통과 08-8-33 계통에서 petri dish 당 20개 정도의 소포자 유래 배 발생이 확인되었다. 높은 소포자 유래 배 발생 계통인 08-8-8 계통과 소포자 유래 배 발생이 되지 않는 08-8-10 계통을 대상으로 화뢰의 크기에 따른 소포자 발달 단계 및 밀도를 조사하였다. 소포자 유래 배 발생 효율이 높은 08-8-8 계통과 소포자 유래 배 발생 효율이 낮은 08-8-10 계통 사이에 화뢰의 구조적 차이는 나타나지 않았다. 그러나 화뢰의 크기가 M 사이즈인 경우에만 주두의 길이가 화엽의 길이보다 더 길었다. 화뢰의 크기와 소포자 수와는 정의 상관을 나타내었으나 S 사이즈의 화뢰인 경우에는 소포자를 관찰할 수 없었다. 화뢰 당 소포자 밀도는 08-8-10 계통보다 08-8-8 계통에서 높게 나타났으며, 화뢰 M 사이즈의 1핵성 후기 소포자 비율은 08-8-8 계통에서는 91.3%, 08-8-10 계통에서는 45.7%로 나타났다.

국내 육성 유채 품종의 소포자 배양을 통한 효율적인 소포자배 생산법의 확립을 위해 '탐미유채'를 대상으로 소포자 배양 시 적당한 꽃봉오리의 크기, 고온처리시간, 배양밀도, 기내 증식조건 등의 배양조건을 구명하고자 실시한 결과, 소포자배 발생은 1핵기말과 2핵기 초기 상태의 소포자가 포함된 3.0~3.5 mm 크기의 꽃봉오리에서 채취한 소포자에서 배발생률이 가장 높았으며, 배지 1 mL 당 약 388개의 소포자 배가 발생하였다. 배양 초기에 32.5℃ 에서 2일간 열처리하였을 때 배발생률이 가장 높았다. 소포자의 배양밀도에 따른 배발생은 5×104 개/mL일 때 가장 높았으며, 배양밀도가 더 이상 높아지면 배발생을 억제하였다. 발생한 소포자배는 0.5mg~cdotL1 NAA와 1mg~cdotL1 BA가 첨가된 MS 고체배지에 치상하여 배양하면 정상적인 신초로 재분화되었고, 발달한 신초를 절취하여 식물성장 조절제가 첨가되지 않은 MS고체배지에서 배양하면 뿌리가 발달하여 정상적인 식물체로 성장하였다.

Composition of nutrient media, flower bud size, cold pretreatment, heat shock stress, and ethylene inhibitor could have marked effects on microspore embryogenesis. No microspore-derived embryos (MDE) were formed when microspores were isolated from radish (Raphanus sativus L.) flower buds of 1.0-2.5 mm in size, whereas MDE were formed with microspores isolated from 2.5-4.5 and 4.5-6.5 mm flower buds. The microspores isolated from 2.5-4.5 mm flower buds showed high embryo yields. When the isolated microspores were washed with Nitsch & Nitsch (NLN) liquid medium containing 130 g‧L-1 sucrose (NLN-13), the yield of MDE increased significantly when comparing with washing using B-5 liquid medium containing 130 g‧L-1 sucrose.Microspores cultured on half strength NLN liquid medium containing 0.05 mgL-1 silver nitrate (AgNO3) produced the most MDE, showing a more than two-fold increase in yield compared to those cultured on medium without AgNO3. A heat shock pretreatment of microspores at 32°C for 24 h gave high-frequency production of MDE when compare to higher or lower temperatures; no MDE were formed at 42.5°C. Microspore viability is known to decrease rapidly with storage; however, in this experiment, microspore viability was maintained for 24 h at 4°C without media. A polyploidy test indicated that 19.7% of the microspore-derived plants were double haploid, other plants were haploid, and chimeras were haploid and diploid. This protocol is thought to be very useful for efficient production of homozygous lines that is critical for the production of radish F1 hybrids

For genetic mapping studies, biparental segregating populations are often useful, however recombination is limited, giving rise to large genomic regions under QTL, and one can only study alelles present in both parents. In Wageninegn UR, a core collection is being developed representing all Brassica rapa morphotypes and geographic origins. As most B. rapa accessions are heterozygous and heterogeneous, we started a project to fix the collection through microspore culture. The resulting Diversity Fixed Foundation Set will be an interesting resource for association mapping studies, which have as advantage that they present the allelec variation present in the collection, and for mapping studies recombination is increased. Nineteen accessions of eight subspecies of Brassica rapa were used for microspore culture to developdoubled haploid lines. Eight morphotypes were represented: 3 Chinese cabbage, 2 Chinese turnip cabbage, 3 Pak choi, 5 Turnip, 3 Broccolleto, 1 Mizuna, 1 Komatsuna and 1 Turnip greenfrom the 19acessions examined, embryos were obtained for 13, representing six subspecies (Komatsuna and Turnip Green had no response). The embryo yields differed significantly between these 13 accessions. We regenerated normal plants from 10 accessions that survived more than 4 weeks in the soil using microspore culture. Nine accessions flowered after 4 weeks vernalization at 5℃ and seeds were harvested from 5 accessions. From a Mizuna, we obtained 3791 seeds from one plant and total 7318 seeds were harvested from 5 accessions representing 4 subspecies(Chinese cabbage, Chinese turnip cabbage, Pak choi, Mizuna). At present, we carry out experiment for obtain more seeds and induce embryos from the other plant materials.

Transient expression profiles for several chimeric β-glucuronidase (GUS) gene constructs were determined in microspore-derived embryos of wheat following microprojectile bombardment. The constructs analyzed consisted of the uidA (GUS) reporter gene driven by six different promoters [Cauliflower Mosaic Virus 35S (CaMV35S), Nopaline synthase (NOS), Mannopine synthase (MAS), Chlorella Mosaic Virus Adenin methyltransferase (AMT), maize Ubiquitin 1 (UBI1), and enhanced 35S (E35S)]. The total numbers of GUS blue spot were determined manually under a dissecting microscope after histochemical staining for GUS. Results suggest that the E35S promoter is the most active and UBI1 promoter is the second active in embryos or embryogenic calli derived from wheat microspore. In addition, by flurometric assay on GUS, the E35S promoter was the best. Therefore, both UBI1 and E35S promoter are suitable for constitutive expression of the gene of interest in microspore-derived embryos of wheat. This information describing promoter functionality in wheat will be important when designing gene constructs for traits modification and when choosing appropriate cultivars for improvement through gene transfer experiments.

The effect of osmotic condition on β-glucuronidase (GUS) transient expression was evaluated in microspore-derived embryos of wheat. Microspore explants were treated on medium containing various mannitol concentrations prior to and post bombardment with plasmid DNA pAHC25 containing uidA gene controlled by maize ubiquitin 1 (UBI1) promoter. GUS expression in the bombarded explants was examined by histochemical and fluorometric assays. Increased GUS expression was observed with mannitol treatment when compared to untreated explants. The histochemical study showed that the number of blue (GUS) foci were the highest in the bombarded explants treated with 0.6 M mannitol medium. The fluorometric assay of bombarded explants also proved 3.5-fold increase in GUS activity with 0.6 M mannitol treatment when compared to without mannitol treatment. These results indicate that 0.6 M mannitol is beneficial for improving transformation efficiency of wheat microspore-derived embryos or embryogenic calli through biolistic transformation.

국내육성 유채품종 간 반수체식물 생산성을 비교하였다. 화아로부터 분리된 소포자를 13%의 sucrose, 0.05mg/ BA 와 0.5mg/ 의 NAA가 첨가된 NLN 배지에서 배양하였다. 반수체 배의 생산에는 유전형이 중요한 요인이였으며, 탐라유채가 가장 높은 반수체 배 생산능을 보여 화아당 176개의 반수체 배가 발생하였으나, 한라유채와 영산유채는 배 생산은 물론 세포분열도 관찰되지 않았다. 발생한 반수체 배를 NLN배지에서 현탁배양하여 Multilobe abnormal embryos를 형성시켰으며, 계속하여 생장조절제가 첨가되지 않은 MS고체 배지에 치상 배양하여 반수체식물체를 유도 하였다. 재생된 반수체 식물체는 성공적으로 순화되었다.

Spikelet proteins expressed at the young microspore stage in rice were separated and analysed by two-dimensional polyacrylamide gel electrophoresis (2DE). The separated proteins were electro blotted onto a polyvinylidene difluoride (PVDF) membrane, and 50 proteins were analyzed by a gas-phase protein sequencer. The N-terminal amino acid sequences of 20 out of 50 proteins were determined. N-terminal regions of the remaining proteins could not be sequenced because of blocking. The internal amino acid sequences of proteins were determined by sequence analysis of peptides obtained by the Cleveland peptide mapping method. Results revealed the presence of the photosynthetic apparatus at rice young microspore stage. Major proteins identified in this study could be used as a marker for various studies on physiological stresses.

An efficient system of rice microspore culture could contribute to the production of genetically modified rice. The microspores were isolated by mechanical or shed methods. The number of microspores per 100 anthers isolated at uninucleate stage was higher than (or similar to) those at binucleate stage in isolation method with pestle or spatular, but microspore divisions were not easily observed on both stages. On the other hand, pollen division in shed pollen culture was observed more frequently at uninuclear than at binuclear stage. Cold pretreatment at 10~circC for 10 days resulted in the best multicellular division to produce microcalli at 12.5% efficiency in shed microspores. Heat shock at 33~circC for one hour before or after pollen shedding enhanced cell division and callus formation. Out of twelve green regenerants, two were haploids and ten were diploids based on the chromosome analysis of root tips. The size of stoma was 12m m in haploids and 15 ~mu~textrmm in diploids determined by scanning electron microscope (SEM).

본 연구는 황기의 생식과 관련하여 자.웅배우체의 발육과정과 화분의 발아 특성을 밝혀서 품종 육성의 기초 자료로 활용하고자 수행한 바, 다음과 같은 결과를 얻었다. 1. 화분의 발육은 화뢰 길이가 3.5mm정도에서 화분모세포가 형성되어 4mm 정도 신장할 때까지 감수분열 및 동형분열하여 4분자기로 되었다. 2. 화뢰의 길이가 10mm정도 되면 성숙화분이 형성되고 12mm정도에서 개약하는 경향이었다. 3. 화분은 4℃ 및 -4℃ 저장시에는 저장후 30일 경에도 발아율이 양호 하였으나 상온(23~28℃ )에서는 저장후 3일부터는 발아력이 3%이하로 저하되었으며 발아속도도 저하되었다. 4.대포자의 형성시기는 소포자(화분)와 거의 비슷한 시기로 화뢰의 크기가 10~12mm에서 완전히 성숙하며, 꽃이 개화 되기전 화분의 미개약시에 자예는 수정 능력이 있는 것으로 나타났다.

Astragalus membranaceus has flowers that are similar to that of the legume family, but shows poor bearing when self-pollination is induced. Thus, this study was carried out observing the ripening procedure of pistils and stamens and development stages of pollen in the context of the birth and growth of the flower. As to the bearing of the flower of A. membranaceus, few pod setting and 13% pod setting were observed when self-pollination is induced by paper-bag covering or artificial pollination treated respectively. The result indicates that A. membranaceus is a cross-pollination plant. A pistil grew faster than a stamen until just before blooming. The flower size was about 17.0mm~times 4.0mm. Pistils and stamens had the same length after flowering. Pollen mother cells passed through meiosis and mitosis when its length reached around 3.5mm, thus creating the tetrade when 4 mm long. Pollen attained full growth when the bud was about 10mm long. An anther was found to tend to dehisce when the length of a bud reached around 12.0mm. As to the shape of pollen, about 70 % were normal. 1% and 30 % were small or empty pollen respectively. The result indicates that pollen of A. membranaceus attains full growth just before anther dehiscence which occurs before blooming while pistils grow faster than stamens until before flowering

소포자초기의 내냉성이 전역물관리와 시비방법에 따라 변동됨을 실증하고 내냉성의 변동기구를 화분발육생리로 부터 검토한 결과를 요약하면 다음과 같다. 1. 소포자초기의 벼의 내냉성은 유화분화기로부터 소포자초기까지의 수온(전역수온) 및 질소(전역질소)에 의하여 현저하게 변동하였고 유화분화기이전과 소포자초기이후의 수온 및 질소에 따른 변동은 거의 없거나 근소하였다. 영화분화기로부터 소포자초기까지는 10일여에 불과하나 벼의 내냉성소질을 경정하는 중요한 시기였다. 2. 전역물관리에 있어 수온은 25℃ 까지 될 수 있는 한 높게. 수심은 10cm까지 될 수 있는 한 깊게 할수록 내냉성이 향상되었으며 이 이상으로 수온상승 및 수심을 깊게하여도 내냉성은 변하지 많았다. 전역수온상승에 따른 내냉성의 향상은 유수가 물로 보호되기 때문이었다. 전역 10cm의 심수관개의 단독효과는 위험기 20cm의 단독효과보다 켰으며 양시기의 심수관개에 따른 냉해방지효과는 상승적이었다. 3. 전역질소의 다량시용에 따른 내냉성의 저하는 엽신의 질소함유율이 어느 한계치를 넘으면 급격히 커졌는데 이 내냉성저하의 변환점에 있어서의 엽신질소 함유율은 일본형에서는 약3.5%, 통일형에서는 약2.5%로 추정되었다. 이 한계엽신질소함유율은 냉해상습지 또는 저온년에 있어서 안전한계시비량을 결정하는 한 지표로 활용될수 있을 것으로 본다. 4. 전역수온상승에 따른 약당충실화분수의 증가는 소포자분화수의 증가에 의한 것이었고 전역질소의 감소에 따른 약당충실화분수의 증가는 소비자의 퇴화에 기인된 것이었으며 충실화분수가 많을수록 임실비율이 높았다.+석회구에서 낮았다 6. 이상과 같은 결과로 미루어 본 실험에 시용된 절대질소량은 많지 않음에도 불구하고 3요소＋유기물구는 유기물 분해에 따른 질소의 추비적 효과가 나타나서 무비, 무질소구에 비해 동할미, 심복백미 등 미립의 외관적 특성을 저하시키고, 단백질함량은 높이고 Mg/KNㆍAmylose를 낮추어 식미를 저하시키는 것으로 해석된다 7. 취급특성으로서의 흡수율, 취급액요드정색도, 취급용액출고형물은 처리간 명백한 경향을 발견할 수 없었다. 품종마다 달랐다.분얼'은 출수기가 빨랐으며, 이들은 3일 내에 모두 출수되어 짧은 기간 내에 균일한 출수와 등숙이 이루어짐으로서 미질 향상에 유리할 것으로 해석된다. 변이의 전분산에 대한 기여율은 각각 16.7%와 56.4%로 산지간 변이가 품종간 변이보다 약 4배 가까이 컸다.종과 이앙기를 조절하여 풍해를 회피하거나 방풍림이나 방풍강을 설치하여 풍해를 경감시키는 것이 수량 생산의 안전성을 향상시킬 수 있을 것으로 생각된다.종 육성 보급이 이루어져야 하고, ~circled3 품수해 발생상습지에서는 벼 피해를 보상할 수 있는 타작물과의 함리적 작부체계 개선 연구가 이루어져야 하고, ~circled4 피해도체의 활용도 증진 연구가 이루어져야 할 것이다. 수 있고 수확량도 크게 증가할 수 있을 것으로 생각된다.들은 진흥과 IR36품종의 중간 영역에 머물고 있었다.mite처리구가 8.6%, 유황첨가구가 5.7~7.4% 증수되었음을 보이었다. 7. 식물체중의 조단백질 함량은 대조구, 5%, 10% 유황첨가구가 3.31~3.50%로서 비슷하였고 15% 유황첨가구는 3.94%,