Map-based cloning is a basic method for identifying the mutated gene in plants. We selected the gametophytic mutant, named as AP-26-09, in activation-tagging pool. Mutant plant showed various kinds of pollen phenotype, such as the different number of nucleus or abnormal shapes. For the map-based gene cloning, we conducted phenotypic analysis of F2 mapping population through the screening of DAPI-stained pollen using fluorescence microscopy. Genomic DNA of F2 plants is prepared from leaves of approximately 1000 plants. In order to define chromosomal region where mutation is located, we designed SSLP markers and performed PCR amplification. In this study, we characterized gametophytic mutant and determined the chromosomal location using map-based approach.

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.

Pollen development in flowering plants is regulated by a comprehensive pattern of genes. One way to produce hybrid rice based on nuclear male sterility is to find out firstly the potential promoters that function specifically in anthers since it is a specific site for transcription initiation and play key roles for the spatial and temporal expression of the genes. To implement this objective, we were selected promoter region of 16 genes based on the expression pattern of microarray and then those were introduced into the promoterless final destination vector which containing the GFP and GUS reporters genes. The resulting twelve vectors were transformed into monocotyledonous rice (Oryza sativa L) and a dicotyledonous Arabidopsis as heterologous system. Minimum 20 plants for each vector were analyzed by histochemical GUS assay at the flowering stage in Arabidopsis. 9 vectors out of 12 vectors constructed were expressed exclusively at the anther, especially in pollen, however one vector exhibited expression in stigma. For rice, T-DNA insertion were confirmed with specific primers in each promoter and GFP region. All T0 transgenic plants contained T-DNA insertion in their genome. This study would provide valuable information for biotechnological application for the induction of male sterility in plants.

Pollen development in flowering plants is regulated by a comprehensive pattern of genes. One way to produce hybrid rice based on nuclear male sterility is to find out firstly the potential promoters that function specifically in anthers since it is a specific site for transcription initiation and play key roles for the spatial and temporal expression of the genes. To implement this objective, we were selected promoter region of 16 genes based on the expression pattern of microarray and then those were introduced into the promoterless final destination vector which containing the GFP and GUS reporters genes. The resulting twelve vectors were transformed into monocotyledonous rice (Oryza sativa L) and a dicotyledonous Arabidopsis as heterologous system. Minimum 20 plants for each vector were analyzed by histochemical GUS assay at the flowering stage in Arabidopsis. 9 vectors out of 12 vectors constructed were expressed exclusively at the anther, especially in pollen, however one vector exhibited expression in stigma. For rice, T-DNA insertion were confirmed with specific primers in each promoter and GFP region. All T0 transgenic plants contained T-DNA insertion in their genome. This study would provide valuable information for biotechnological application for the induction of male sterility in plants.

교배효율의 증대를 위해서는 정상화분의 확보가 선행되어야하며 화분의 활성을 파악할 필요가 있다. 본 연구에서는 화분의 활성을 확인하기 위해 여러 가지 염색액을 사용하여 광학현미경하에서 관찰하였다. Aceto-carmine과 Alexander's 염색액을 사용한 실험에서 비슷한 정상화분율을 얻을 수 있었고 FCR염색으로 관찰한 결과 살아있는 화분만 정확히 선별해 주기에 가장 낮은 정상화분율을 얻을 수 있었다. TB는 화분의 상태에 따라 달랐으나 Aceto-carmine과 Alexander's 염색액을 사용한 실험에서의 결과와 비슷한 양상을 보였다. 화분 관찰의 경험이 별로 없는 실험자도 쉽게 접근할 수 있는 염색액은 Alexander's 이었다. 정확한 화분의 활성을 조사하는 데에는 FCR이 필수이다. 형광현미경 장치 없이 정상과 비정상화분의 구별이 용이한 Alexander's 염색액으로 관찰한 자료를 바탕으로 살아있는 정상화분율의 예견이 가능하나 정확한 임성을 확인하려면 추가로 FCR test를 해야 한다.