BIOLOGICAL AND MOLECULAR ANALYSIS OF INDUCTION MECHANISM FOR SOMACLONAL VARIAION IN TOMATO

当番茄栽培品种‘Ponderosa’的叶段用携带双元载体pRi和pBI 121/sGFP的发根农杆菌MAFF 07 -20001接种时，不定根从在所述段的边缘形成的愈伤组织发育。获得了在蓝光下显示绿色荧光并在无蔗糖培养基上剧烈伸长而不损失绿色荧光的原始根。它们很容易从相同节段上的非荧光根中区分出来。通过聚合酶链反应和Sourhem杂交证实了sGFP和rol C基因成功整合到番茄根的染色体中。本方法使我们能够评估毛状根的形成，而无需继代培养，分离和DNA分析。该方法在日本所有的商业品种（42个品种）和14个番茄育种系上进行了测试。除了两个繁殖系外，所有繁殖系都产生了毛状根。因此，本发明的方法可用于毛状根生产 ...更多信息 番茄酱。利用RT-PCR技术对番茄愈伤组织中单个细胞的基因表达进行了原位检测。在光学显微镜下观察，用微量移液管从一个细胞中取出细胞质，直接用于RT-PCR，然后进行巢式PCR。这种去除胞浆内容物的方法阻止了基因组DNA引入RT-PCR，并且当使用含内含子的基因作为PCR靶时，仅扩增内含子拼接产物。此外，无内含子基因的转录可能通过使用针对靶基因的混合引物同时追踪含内含子基因和无内含子基因来检测。本研究表明，一些受刺激激活的基因，如CHI 3和TLC 1-反转录转座子长末端重复序列，在番茄愈伤组织细胞中组成型转录。采用单细胞RT-PCR技术对番茄叶片毛状体细胞中的基因表达进行了原位检测。使用光学显微镜用微量移液器去除细胞质，并直接用于RT-PCR，然后进行巢式PCR。两个含内含子的基因，甘油醛3-磷酸脱氢酶基因和质膜H^+-ATPas基因在该组织中持续表达，因此被用作指示物，因为很容易检测到剪接产生的较短的PCR产物。此外，吸取细胞内容物的方法可以有效地防止基因组DNA的污染，从而避免在RT-PCR和随后的巢式PCR过程中，缺失内含子基因的相应基因组DNA序列的错误扩增。因此，本技术可应用于番茄叶片的单个毛状体细胞，以直接检测其响应于化学和物理刺激的基因表达。少

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 … More 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