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

用携带pRi和pBI121/sGFP二元载体的根生农杆菌MAFF07-20001接种番茄‘黄豆’叶片，叶片边缘愈伤组织形成不定根。原始根在蓝光照射下显示绿色荧光，在无激素培养基上生长旺盛，且绿色荧光无损。它们很容易与同一片段上的非荧光根区分开来。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染色体上。本方法无需传代培养、分离和DNA分析，即可对毛状根的形成进行评价。该方法在日本所有商品番茄品种（42个品种）和14个番茄育种品系上进行了试验。除了两个育种系外，所有育种系都产生了毛状根。因此，该方法可用于番茄毛状根的生产。采用RT-PCR技术对番茄愈伤组织聚集体中单个细胞的基因原位表达进行检测。取一个细胞的细胞质，用微管在光镜下观察，直接进行RT-PCR，然后进行巢式PCR。这种去除细胞质内容物的方法阻止了基因组DNA导入RT-PCR，并且当含有内含子的基因作为PCR靶点时，只能扩增内含子剪接的产物。此外，利用混合引物对目标基因同时追踪含内含子基因和不含内含子基因，可能检测到无内含子基因的转录。本研究表明，刺激激活基因CHI3和tlc1 -反转录转座子长末端重复序列在番茄愈伤组织中存在组构性转录。采用单细胞RT-PCR技术原位检测番茄叶片毛状体细胞基因表达。光镜下用微移液管除去细胞质，直接进行RT-PCR，然后进行巢式PCR。两个内含子基因，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，由于易于检测剪接产生的较短尺寸的pcr产物，因此可以作为指示物。此外，吸去无核细胞内容物可以有效防止基因组DNA被污染导致在RT-PCR及后续的巢式PCR过程中缺失内含子基因对应的基因组DNA序列。因此，该技术可应用于番茄叶片单个毛状细胞，直接检测其在化学和物理刺激下的基因表达。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