• Journal of Life Science
• /
• 제15권6호
• /
• pp.1034-1036
• /
• 2005

Chinese cabbage (Brassica campestris ssp. napus var. pekinensis Makino) seeds/seedlings were transformed via vacuum-infiltration with recombinant Agrobacterium tumefaciens LBA4404 cells. The agroinfiltration method was determined to be unsuccessful for Chinese cabbage transformation during the analysis of hepatitis B surface antigen expression by ELISA. However, treatment of sodium hypochlorite solution, prior to agroinfiltration, to pregerminated or germinating 1 day- or 2 days-old seeds was proven effectively to enhance transformation efficiency, suggesting that chemical wounding caused by sodium hypochlorite reaction might facilitate Agrobacterium infection and, therefore, transient gene expression in Chinese cabbage sprouts.

• KSBB Journal
• /
• 제21권6호
• /
• pp.489-492
• /
• 2006

For molecular breeding purpose, genetic transformation of Brassica napus cultivars has been extensively performed using Agrobacterium method. B. napus cv. napus, one of major oil crops, can be transformed via Agrobacterium-based method. We demonstrated that Agrobacterium-mediated transformation via vacuum infiltration slightly worked for the seedlings of B. napus cv. napus according to fluorometric GUS enzyme analysis. In contrast, transformation efficiency was highly enhanced when the seedlings, prior to agroinfiltration, were treated with sodium hydrosulfite solution as a chemical wounding agent. GUS gene expression in transformed seedlings that was confirmed by RT-PCR suggests their usefulness for the development of transient expression system.

• Journal of Plant Biotechnology
• /
• 제7권4호
• /
• pp.219-223
• /
• 2005

We investigated chemical-wounding effect on Agrobacterium-mediated Chinese cabbage transformation via vacuum infiltration. Pre-germinated or germinating Chinese cabbage seeds were infiltrated with Agrobacterium tumefaciens LBA4404 cells carrying either GUS gene (pBI121) or hepatitis B virus surface antigen DNA (pBIHBsAg). Prior to agroinfiltration process, the seeds were soaked in sodium hydrosulfite (SHS) solution or just in sterile water as a control. Comparative transformation efficiency was determined by both of histochemistry and ELISA. We could demonstrate that SHS solution treatment especially to 1-day or 2-days old germinating seeds efficiently improved transformation process, and therefore, transient expression level. This strongly indicated that Agrobacterium infection could be facilitated indeed by SHS-causing wounds on Chinese cabbage seeds.

• Plant Biotechnology Reports
• /
• 제2권1호
• /
• pp.1-6
• /
• 2008

Cotyledonary leaves of tomato cv. Megha were transformed with the hepatitis B virus 's' gene, which encodes surface antigen. Six plant expression cassettes (pHBS, pHER, pEFEHBS, pEFEHER, pSHER and pEFESHER) were used to assay the possible expression levels by agroinfiltration. The maximum transient expression level of 489.5 ng/g D.W. was noted in pEFEHER-infiltrated cotyledonary leaves. Transgenic tomato plants with pEFEHBS and pEFEHER expression cassettes were regenerated and characterized by molecular analysis. The expression of the antigen in the fruits was confirmed by RT-PCR and ELISA analysis. This is the first report on the expression of hepatitis B surface antigen in tomato.

• Journal of Applied Biological Chemistry
• /
• 제48권4호
• /
• pp.229-230
• /
• 2005

• Applied Biological Chemistry
• /
• 제49권4호
• /
• pp.349-351
• /
• 2006

• The Plant Pathology Journal
• /
• 제33권6호
• /
• pp.608-613
• /
• 2017

The full-length sequence of a new isolate of Apple chlorotic leaf spot virus (ACLSV) from Korea was divergent, but most closely related to the Japanese isolate A4, at 84% nucleotide identity. The full-length cDNA of the Korean isolate of ACLSV was cloned into a binary vector downstream of the bacteriophage T7 RNA promoter and the Cauliflower mosaic virus 35S promoter. Chenopodium quinoa was successfully infected using in vitro transcripts synthesized using the T7 promoter, detected at 20 days post inoculation (dpi), but did not produce obvious symptoms. Nicotiana occidentalis and C. quinoa were inoculated through agroinfiltration. At 32 dpi the infection rate was evaluated; no C. quinoa plants were infected by agroinfiltration, but infection of N. occidentalis was obtained.

• Plant Biotechnology Reports
• /
• 제5권3호
• /
• pp.273-281
• /
• 2011

Diverse epigenetic phenotypes are frequently found during research on transgenic plants. To understand the factors underlying such diversity, hundreds of independent 35S-GFP transgenic N. benthamiana plants were analyzed. The diverse GFP-expression phenotypes of the transgenic plants were classified into three major types based on the GFP expression patterns and their response to 35S-GFP agroinfiltration: steady-green, silenced and non-uniform phenotype. The non-uniform phenotype was further sub-divided into five minor phenotypes: variegated, red-dropped, on-silencing, partitioned and misty, according to the distribution of GFP expression on the leaves. Many of transgenic plants continuously generated diverse phenotypes over several generations despite the transgene identity. Such epigenetic GFP phenotyping was found to be the result of spontaneous transgene silencing mediated by either or both of post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS). This finding was verified by the detection of 21- and 24-nt small interfering RNA (siRNA) molecules, and DNA methylation in the transgenic plants that showed repeated epigenetic variation. Agroinfiltration demonstrated that irregular distribution of GFP on a leaf was the result of erratic transgene silencing, and the technique also proved to be a rapid and effective method for selecting fully silenced plants within 3 days. Furthermore, two novel phenotypes described are potential materials for in-depth investigations into the genes and mechanisms responsible for spontaneous transgene silencing.

• Horticultural Science & Technology
• /
• 제31권2호
• /
• pp.246-254
• /
• 2013

Five isolates of Tomato yellow leaf curl virus (TYLCV) collected from various regions of Korea were amplified using PCR and determined the sequences of full-length genome, respectively. The PCR-amplified DNA of each TYLCV isolate was introduced into a binary vector to construct infectious clone containing 1.9 copies of the corresponding viral genome. Various cultivars and breeding lines of tomato were inoculated with Agrobacterium tumefaciens harboring infectious clone of each TYLCV isolate to assess resistance against TYLCV. Susceptible cultivar 'Super-sunread' revealed typical yellowing and narrowing of the upper leaves. In contrast, breeding linesTY12, GC9, GC171, and GC173, which contained the TY-1 and/or TY-3 genes that confer resistance against TYLCV in nature, were completely symptomless, suggesting that the lines were resistant to challenging TYLCV isolates. Symptoms of TYLCV in susceptible tomato cultivars are significantly different from those of TYLCV in the resistant tomato cultivars at 30 days after agroinfiltration. Although genomic DNAs of TYLCV were detected from the breeding lines TY12, GC9, GC171, and GC173 using real-time PCR analysis with specific primers, levels of TYLCV DNA accumulation in the resistant breeding lines were much lower than those of TYLCV DNA accumulation in susceptible tomato cultivars. Similar symptom severity and levels of TYLCV DNA accumulation were observed from TYLCV infections mediated by Bemisia tabaci in the resistant and susceptible tomato cultivars. Concentration of agrobacterium did not affect the response of tomato cultivars against TYLCV inoculation. Taken together, these results suggest that TYLCV inoculation via agroinfiltration is as effective as inoculation through Bemisia tabaci and is useful for breeding programs of TYLCV-resistant tomato.

• Applied Biological Chemistry
• /
• 제51권4호
• /
• pp.342-343
• /
• 2008