| Research Abstract |
When leaf segments of a tomato cultivar 'Ponderosa' were inoculated with Agrobacterium rhizogenes MAFF07-20001 carrying the binary vectors pRi and pBI121/sGFP, adventitious roots were developed from calli formed at the edges of the segments. Primordial roots that showed green fluorescence under blue light and elongated vigorously on hormone-free medium without loss of the green fluorescence were obtained. They were easily distinguishable from the non-fluorescing roots on the same segments. Successful integration of the sGFP and rol C genes into the chromosome of tomato roots was confirmed by polymerase chain reaction and Sourhem hybridization. The present method enables us to evaluate the hairy root formation without subculture, isolation and DNA analysis. This method was tested on all of commercial cultivars available in Japan(42 cultivars) and 14 breeding lines of tomato. All but two breeding lines produced the hairy roots. Thus, the present method is useful for hairy root production
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in tomato. RT-PCR was used to detect gene expression in situ in single selected cells from tomato callus aggregates. The cytoplasm from one cell was removed with a micropipette viewed under a light microscope and used directly for RT-PCR, followed by nested PCR. This method of removing cytosolic contents prevented the introduction of genomic DNA into the RT-PCR, and only intron-spliced products were amplified when intron-containing genes were used as PCR targets. In addition, transcription of the intron-free gene was possibly detected by simultaneously tracing the intron-containing and intron-free genes using mixed primers for the targeted genes. The present study indicated that some stimuli-activated genes, such as CHI3 and TLC1-retrotransposon long terminal repeat, were constitutively transcribed in tomato callus cells. A single-cell RT-PCR was conducted to detect gene expression in situ in pinpointed trichome cells of tomato leaves. The cytoplasm was removed with the micropipette using a light microscope and directly used for RT-PCR, followed by nested PCR. Two intron-containing genes, glyceraldehydes 3-phosphate dehydrogenase gene and plasma membrane H^+-ATPas gene were constantly expressed in this tissues and therefore used as the indicator, because of easy detection of shorter-size PCR-products produced by splicing. In addition, the sucking of nucleus-free cellular contents was effective to prevent contamination of genomic DNA led to miss-amplification of corresponding genomic DNA sequences of the intron-less genes in the process of RT-PCR and subsequent nested PCR. Thus, the present technique could be applicable to single trichome cells of tomato leaves for directly detecting their gene expression in response to chemical and physical stimulation. Less
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