The AtBI-1 gene encoding the Arabidopsis thaliana Bax inhibitor was introduced into Japonica cultivars of rice (“Ilmi”) by Agrobacterium-mediated transformation, and a large number of transgenic plants were produced. The neomycin phosphotransferase II (NPTII) gene was used as a selectable marker. The activity of neomycin phosphotransferase could be successfully detected in transgenic rice calluses. Introduction of the AtBI-1 gene was also confirmed by PCR using AtBI-1 specific oligonucleotide primers in regenerated plants. Stable integration and expression of the AtbI-1 gene in plants were confirmed by GFP analysis.
Rice is the one of the most important cereal crops in the world because it has a more complex nutritional content than other cereals, including proteins, carbohydrates, vitamins and minerals. According to Kiple and Ornelas [
Japonica rice (Oryza sativa L.) cv. “Ilmi” was used as the explant in this study. Dehulled mature seeds were soaked in 70% ethanol with vigorous shaking for approximately 1 min, washed in sterile distilled water three times, and then incubated in sterilization liquid (sterile distilled water containing 1% NaOCl) for 30 minutes with 120 rpm. The samples were then washed with sterile water three times, after which they were dried on sterile filter paperfor an hour in room temperature and pre-cultured on PCI (
Agrobacterium tumefaciens strain LBA 4404:pBin-AtBI- 1-GFP was cultured in PB (
which they were grown on PSRmedia at 30˚C ± 2˚C for 1 month under continous light. Aftershoot sappeared from the calli, the samples were (hereafter referred to as putative transformants) transferred to test tubes containingat 28˚C PSR (
Leaves of putative transformants were harvested and transferred into acclimatization medium, after which genomic DNA was extracted using a genomic Dneasy Plant kit (Qiagen). Polymerase Chain Reaction (PCR) analysis was then performed usingspecific primersfor the kanamycin resistance gene (forward: 5’-ATGGATGCG TTCTCTTCCTT-3’; reverse: 5’-AACAGGATTATCAT TTTCCTT-3’). PCR was conducted by subjecting the samples to the following conditions: predenaturation at 94˚C for 2 minutes, followed by 30 cycles of denaturation at 94˚C for 30 seconds, annealing at 56˚C for 30 seconds and extension at 72˚C for 30 seconds and then post-extension at 72˚C for 4 minutes. The PCR products were then subjected to electrophoresis on 1% agarose gel and visualized by ethidium bromide staining.
The target protein, AtBI-1, was observed by confocal laser scanning microscopy (LSM700, Carl Zeiss, German). Visualization of GFP in leaves following the induction of AtBI-1 expression revealed that GFP showed cytoplasmic localization that was indistinguishable from that observed for the GFP-only control. In contrast, AtBI-1 expression was predominantly in the cell.
Callus induction is an important stage in the transformation process to produce embryogenic callus as explant transformation. Callus formation in rice (Oryza sativa L.) cv Japonica “Ilmi” cannot be distinguished from callus formation in other plants. This process begins with the formation of an embryogenic callus, after which globular somatic embryogenesis occurs. At this stage of callus induction, the embryogenic callus is very important as an effective medium for genetic transformation either through particles or Agrobacterium [
It is important to optimize infection of plant cells by Agrobacterium to ensure transformation of the callus. The optical density of the Agrobacterium was used to ensure stable and efficient transformation potency. This is because high concentrations of Agrobacterium can produce overgrowth on medium during co-cultivation, while low concentrations can reduce transformation efficiency. In this study, we used 0.1 OD600 for infection. Rice is not a dicotyledone plant, so it cannot produce phenolic compounds as stimuli in response to infection. In the mechanism of infection, phenolic compounds act as the signaling mechanism by which bacteria express virulence genes (vir A and vir G) and sugar molecules serve as coinducers. Sugar was responded by proteins, ChvE, which is encoded by a gene on chromosome Agrobacterium. In the presence of sugar, low concentrations of phenol are more effective for induction of genomic plants [
Following infection, co-cultivation was conducted for 4 days at 30˚C and then at 28˚C for 3 days under continuous dark conditions. Incubation temperature was shown to play an important role in management of the Agrobacterium growth and optimizing infection of the plant genome. Agrobacterium can grow well at 28˚C; however, in this study, the use of 30˚C for the first temperature did not impact the infection. Nijmona and Lamparter [
During the selection and regeneration stage it was important to kill Agrobacterium tumefaciens that had infected the callus. Therefore, we rinsed infected calluses with sterilized water and added carbenecilin to kill the Agrobacterium.
In general, the results showed that shoots appeared approximately one month after washing, and it would be occur the amount of putative plant, but, the total of callus infected would decrease because kanamycin resistance.
The uninfected plants also died because kanamycin antibiotic could attack the plant cells (
The leaves were harvested from 4 putative plants following the first infection, one putative plant following the
*Infection 1: 25 days inducing callus; Infection 2: 35 days inducing callus; Infection 3: 45 days inducing callus.
second infection and one putative plant after the third infection. And that, we seperated the plant contained more than one plant and we got 20 putative plants, consist of: 16 putative plants first infection, a putative plant second infection and 3 putative plants third infection. PCR analysis showed that among 20 putative plants, there were three transformed plants (
To investigate the potential effects of theAtBI-1-GFP gene, we constructed the vector pBin-AtBI-1-GFP and introduced it into rice. GFP (Green Fluorescent Protein)
*Infection 1: 25 days inducing callus; Infection 2: 35 days inducing callus; Infection 3: 45 days inducing callus. **a: green plant, b: albino plant, c: white-green plant.
is a common maker of transformation; therefore, the leaves of three transformant plants were tested for GFP expression. The results revealed that the leaves were loaded with GFP, and GFP imaging showed a stronger signal in transgenic plants than non-transgenic plants (
This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ00950503), Rural Development Administration, Republic of Korea. We are grateful to Dr. Hirofumi Uchimiya in Institute of Molecular and Cellular Biosciences, The University of Tokyo.