ISSN : 2287-5174(Online)
Optimization of Agrobacterium-mediated Transformation in Japonica-type Rice Oryza sativa L. cv. Dongjin for high Efficiency
Rice is one of the most important cereal crops in the world, and it feeds billions of the world’s population. Recent global climate change has altered environmental conditions important for agriculture, including rice production, and has compromised crop production and food security (Lobell et al. 2011). However, it may be possible to overcome this issue through biotechnology. Rice genome sequencing has been completed, and there are many rice genetic resources such as several cultivars, mutants and mapping populations (Sasaki et al. 2001; Hirochika et al. 2004). Rice is also important as a model system for functional identification of genes in monocots through genetic study.
For many years, scientists have tried to develop transformation systems using different approaches such as protoplast transformation, particle bombardment transformation and Agrobacterium-mediated transformation methods (Hayashimoto et al. 1990; Chan et al. 1993; Christou, 1995). However, the development of reliable and efficient transformation systems remains an important goal in molecular biology and biotechnology of rice. The advantages of Agrobacterium-mediated gene transfer over other methods include the high efficiency of transformation, the ability to transfer relatively large segments of DNA, and a relatively short transformation duration (Gelvin, 2003). Thus, Agrobacterium-mediated methods have been used the most popular in recent years.
For Agrobacterium-mediated transformation of rice, different explant types such as mature seed-derived calli, immature embryo-derived calli, and shoot apices were tested for efficient transformation (Hiei et al. 1994; Toki et al. 2006). It was reported that 3-week-old (secondary) calli generated from mature seeds were competent for Agrobacterium-mediated transformation (Hiei et al. 1994). This method and modifications of it have been used by many scientists (Vijayachandra et al. 1995; Sallaud et al. 2003; Saika and Toki, 2010). However, continuous callus culture for at least 3 months before obtaining transformants can lead to undesired genomic changes (somaclonal variation). This somaclonal variation is one of the obstacles to performing phenotypic analysis of transgenic rice for functional identification of genes. One-day-old (primary) calli generated from scutellum tissue have also been reported to be competent for Agrobacterium-mediated transformation, and a transformation method was developed based on that (Toki et al. 2006). This tissue culture system is one of the most efficient transformation methods with the shortened transformation duration and decreased undesirable genomic changes (somaclonal variation occurrence). However, transformation efficiency is dependent on the cultivar used, and there are many rice cultivars. Thus, Agrobacterium-mediated transformation should be optimized for each cultivar. Here, we have improved transformation efficiency by addition of the endosperm removal step, changing the Agrobacterium strain and media composition. We also analyzed the copy number of transferred genes and morphological changes in transgenic plants when this transformation method was used. This study will provide valuable information to rice researchers.
MATERIALS AND METHODS
Plant materials and callus generation
Oryza sativa ssp. Japonica cv. Dongjin was used in this study. Mature seeds were dehulled and pre-rinsed in 70% ethanol with vigorous shaking for 1min. Dehulled seeds were incubated for 40 min with sterilization buffer (2.5% NaClO solution containing 0.02% Tween 20) and washed with sterile distilled water four times. Seeds were pre-cultured on 2N6 medium at 28°C under continuous dark condition for 5 days. Endosperm was carefully removed by sterilized forceps, and then calli were harvested. The composition of culture media is described in Table 1.
Table 1. List of Meida used in this study.
Agrobacterium preparation and infection
Agrobacterium tumefaciens strains LBA4404 and EHA105 with the pGA2897 binary vector were used for plant infection. Agrobacterium was grown on AB medium including 5 mg/L tetracycline and 50 mg/L rifampicin at 28°C for 3 days (Chilton et al. 1974). Cultured cells were suspended in AAM liquid medium with 100 μM acetosyringone and adjusted to 0.1 OD595 and then diluted to 0.01 and 0.001 OD595. For Agrobacterium infection, pre-cultured calli were inoculated with Agrobacterium suspension for 2 min in a 50 mL conical tube and excess bacteria were removed with sterilized filter paper.
Co-culture and selection of transformed embryogenic callus
Calli were transferred to filter paper which had been previously soaked with 0.5 ml AAM medium and placed on 2N6-AS medium. Infected calli were co-cultured on 2N6-AS medium at 25°C in the dark for 3, 5, or 7 days. Next, cotyledons, roots and endosperm tissues were removed from embryogenic callus in order to obtain intact callus tissues. Calli were then incubated on 2N6-CH medium containing 2,4-D, hygromycin and cefotaxime (2 mg/L, 40 mg/L and 250 mg/L, respectively) at 28°C in the dark for one or two weeks.
Regeneration of transgenic plants
Transformed calli were transferred and cultured on regeneration medium (MSR-CH medium) for 8 weeks in a growth chamber at 28°C under conditions of a 16 h light/8 h dark cycle. White and actively growing calli were transferred to fresh regeneration medium every two weeks. Transformants arising from the calli were rinsed with running water on 60 days after sowing. Transformants were transferred to 1% hyponex solution (Hyponex, Japan) in a 50 ml conical tube for 1 day in growth condition for regeneration. Transformants were then acclimated to green house condition for 5 days and plantlets were grown in the rice paddy or green house to harvest seeds.
PCR analysis and Southern blot analysis
Leaves of rice were harvested and genomic DNA was extracted from the wild type and transformants using a genomic DNA prep kit (SolGent, Korea). Polymerase chain reaction (PCR) analysis was performed with specific primer for the hygromycin resistance gene (Forward primer; 5’-ATGAAA AAGCCTGAACTCAGGGC-3’ and Reverse primer; 5’-CT ATTCCTTTGCCCTCGGACGAG-3’) using the following cycling parameters: denaturation at 94°C for 3 min, followed by 25 cycles of 94°C for 30 sec, 58°C for 30 sec, 72°C for 1 min, and a final extension at 72°C for 5 min. PCR products were separated by electrophoresis on a 1% agarose gel and visualized by ethidium bromide staining.
Genomic DNA was digested with BamHI and HindIII and separated by electrophoresis on a 1.2% agarose gel, then transferred to an Amersham Hybond-C membrane (GE Healthcare, UK). Hybridization was performed at 65°C overnight with full-length cDNA of hygromycin phosphotransferase that was labeled using a random oligonucleotide priming kit (GE Healthcare, UK). The membrane was washed with 2X SSC/ 0.1% SDS for 15 min and rewashed with 1X SSC/0.1% SDS for 15 min at 65°C. The washed membrane was exposed to a BAS screen (Fujifilm, Japan). Images were captured with a Personal Molecular Imager (BIO-RAD, USA) and processed with Adobe Photoshop software (Adobe Systems, USA).
Agrobacterium strains and concentrations affect transformation efficiency in japonica-type rice cultivar Dongjin
We generated transformants using scutellum-derived calli from dehulled mature seeds by following the tissue culture procedures shown in Figure 1: embryogenic callus induction, co-cultivation, selection of hygromycin-resistant calli, regeneration and shoot induction (Figure 1). To determine the callus formation efficiency in Dongjin, 100 dehulled mature seeds were sterilized with NaClO and inoculated onto 2N6 media for 5 days at 28°C under dark condition. The callus formation rate was about 80% under our experimental conditions. In this study, we used 60 calli (for three replications) derived from the scutellum of mature seeds as the starting material. In order to quantify transformation efficiency, we recorded the number of hygromycin-resistant calli during the transformed callus selection step and the number of hygromycin-resistant plantlets upon shoot induction before planting on soil.
Fig. 1. The overview of tissue culture processes to develop transgenic rice from scutellum-derived calli. (A) Germination and embryogenic calli induction from dehulled mature seeds. (B) Co-cultivation with A. tumefaciens after removal of endosperm from embryogenic calli. (C) Selection for hygromycin-resistant calli. (D) Shoot induction and shoot elongation from vigorously growing calli. (E) Acclimation and growth of transgenic plants in green house.
Different strains of Agrobacterium have been used for infection, and we tested two different strains: LBA4404 and EHA105. Plant binary vector pGA2897 was introduced to these stains and used for this study. We first examined the effects of the strains on transformation efficiency. For infection, LBA4404 and EHA105 were grown on AB medium with antibiotics and then suspended and adjusted to 0.01 OD595 in AAM medium. These strains were introduced to pre-cultured scutellum-derived calli in conical tubes for 2 minutes and transformants were generated by following tissue culture processes as shown in Figure 1. When LBA4404 was used for infection, the percentage of hygromycin-resistant calli was over twice that obtained when EHA105 was used for infection (Figure 2A). When the percentage of hygromycin resistant plantlets was compared, LBA4404 again gave rise to twice as many as EHA105 (Figure 2B). Given that the LBA4404 strain showed about two-fold higher transformation efficiency compared to the EHA105 strain, we used the LBA4404 strain for further studies.
Agrobacterium cell concentration for infection is an important element for stable and efficient transformation because high concentrations of cells can cause overgrowth of Agrobacterium on the medium and low concentrations of cells reduce the transformation efficiency. Therefore, in order to test which concentrations of Agrobacterium are suitable for stable and high efficiency transformation in Dongjin, cell concentrations were adjusted to 0.1, 0.01, or 0.001 OD595 in AAM medium. These different concentrations of cells were used to infect pre-cultured scutellum-derived calli. The percentage of hygromycinresistant calli obtained from each concentration of Agrobacterium cells were not markedly different (Figure 2C). However, the percentage of hygromycin-resistant plantlets showed a small effect of Agrobacterium cell concentration, with 0.01 OD595 giving rise to the most transformants (Figure 2D). Thus, a cell concentration of 0.01 OD595 is preferable for obtaining transformants with the highest efficiency in Dongjin using Agrobacterium strain LBA4404.
Fig. 2. Agrobacterium strain and cell density influences on transformation efficiency of rice. (A and B) Comparison of Agrobacterium strains on transformation efficiency. LBA4404 or EHA105 cells (OD595=0.1) were inoculated onto the scutellum-derived calli (pre-cultured calli generated from the scutellum tissue of dehulled mature seeds). (C and D) The effect of Agrobacterium cell density on transformation efficiency. Cultured Agrobacterium LBA4404 was adjusted to an OD595 of 0.001, 0.01, or 0.1, and inoculated onto pre-cultured calli. Transformation efficiency was calculated in terms of the percentage of hygromycin-resistant calli produced on 2N6-CH medium (A and C) or the percentage of regenerated plantlets on regeneration medium before planting on soil (B and D). Error bar presents the standard error.
The effect of endosperm removal before Agrobacterium infection on transformation efficiency
Several research groups have used calli derived from scutellum tissue of mature seeds with endosperm for Agrobacterium-mediated transformation. However, endosperm can cause contamination during tissue culture. We tried to eliminate any possibility of contamination by removing the endosperm from the scutellum tissue with forceps before co-culture. However, this endosperm removal step is laborious and different from the methods used by other research groups. Therefore, we examined whether the endosperm removal of mature seed can have an effect on transformation efficiency in Dongjin. We performed tissue culture with or without endosperm removal from scutellum-derived embryogenic calli before co-culture (Figure 3). Approximately 25% fewer hygromycin-resistant calli were produced when Agrobacterium-mediated transformation was performed with endosperm, compared to callus induction after endosperm removal (Figure 3C). Interestingly, the hygromycin-resistant plantlet rate showed almost two-fold difference between the two treatments (Figure 3D). This result suggests that endosperm removal is an important step in terms of transformation efficiency.
Fig. 3. Endosperm removal from scutellum-derived calli increases transformation efficiency of rice. (A) Photograph of scutellum- derived embryogenic calli from dehulled mature seeds with seed endosperm. (B) Photograph of scutellum-derived embryogenic calli from dehulled mature seeds without seed endosperm. (C and D) The effect of endosperm removal on transformation efficiency. Agrobacterium was inoculated onto scutellum-derived embryogenic calli with or without endosperm. Where indicated, endosperm from dehulled mature seeds was eliminated using sterilized forceps. Transformation efficiency was calculated by measuring the hygromycin-resistant callus production rate on 2N6-CH medium (C) or by counting regenerated plantlet on regeneration medium before planting on soil (D). Error bar presents standard error.
In an effort to reduce the total tissue culture time, we also tested the effects of the co-culture period and the time of callus induction on 2N6-CH medium. Transformation efficiencies were compared after 3, 5, or 7 days of co-culture and one or two weeks of callus induction on 2N6-CH medium, but shortening the time of co-culture time (to 3 or 5 days) and callus induction time (to one week) caused a 10% decrease in efficiency (Data not shown). However, if seeds are plentiful enough and transformation efficiency is not vitally important, at least one week can be saved.
Evaluation of primary transformants with PCR and Southern blot analysis, germination test and morphology observation of transgenic plants
After regeneration and shoot induction, plantlets were cultured on soil in a greenhouse for 1 month, and then transplanted in the field. During the adaptation period in the greenhouse, genomic DNA from leaves of regenerated plantlets was extracted and PCR analysis was performed with hygromycin phosphotransferase (Hph)-specific primers to examine whether T-DNA was inserted into the rice genome. As shown in Figure 4B, when genomic DNA isolated from non-transgenic plants was used as template, the PCR product was not amplified. However, in all transformants, Hph gene was clearly amplified (Figure 4). Next, to evaluate how many T-DNA copies were inserted into the rice genome and whether these regenerated plantlets were independent lines or not, Southern blot analysis was carried out using the Hph gene as a probe. As shown in Figure 4, no signal band was found in wild type plant and all of the transformants showed signal bands of different sizes, indicating that those were independently derived plantlets. In most transformants, two copies of the T-DNA were inserted into the genome (Figure 4).
Fig. 4. Validation of T-DNA insertion in transformants. (A) Vector map. (B) The Hpt gene in the T-DNA of transformants was detected by PCR analysis using gene-specific primers. Genomic DNA was extracted from non-transgenic (Dongjin) and transgenic plants. (C) T-DNA copy number in transformants was estimated by Southern blot analysis. Genomic DNA was digested with BamHI and HindIII and hybridized with the Hpt gene as probe.
T0 (plants transformed) plantlets adapted in the greenhouse were transplanted and cultured in the field, and we investigated the morphology and fertility of the transformants. Transformants harboring pGA2897 vector did not show any severe defects in fertility, tiller number, or leaf height (Data not shown). After harvesting seeds, we tested the hygromycin resistance of T1 (next generation of T0 plant) seeds (50 ug/ml in water) using 50 seeds for each independent plantlet (Figure 5A). Germination rates of T1 generation seeds were more than 70% (Figure 5B). Ten germinated T1 seeds of three independent lines were planted on soil and the developmental phenotypes were observed. Most of the transformants showed similar development phenotypes each other (Figure 5C).
Fig. 5. Segregation of hygromycin-resistant plants and morphology of transformants. (A)Hygromycin-resistant progeny was selected on solution containing hygromycin for 5 days. (B)Estimation of hygromycin-resistant progeny from T1 generation. 36 seeds of each transformant (T1 generation) were incubated on solution including hygromycin for 5 days and seeds showing leaves were counted to determine the germination rate. (C)Photograph of transgenic rice grown for three months. WT; non-transgenic plant (Dongjin), 1-3; T1 generation of transgenic plants.
Japonica-type rice Dongjin is one of the most popular cultivars for research in Korea. Two Korean research groups have established T-DNA insertion mutant pools and Ac/Ds-mediated mutant pools using this cultivar (Jeon et al. 2000; Park et al. 2007). These are the most widely utilized mutant pools for genetic studies in rice research in the world. To ascertain biological functions with these mutants, complementation or over-expression via transgenic lines is necessary. However, conventional Agrobacterium-mediated rice transformation systems using secondary calli require 14 - 16 weeks for establishing transgenic plantlets and somaclonal variation caused during long tissue culture through the Tos17 retrotransposon is thought to be problematic (Hirochika et al. 1996). Therefore, the strategy used for generation of transgenic rice is important. Recently, Toki et al. reported a short-term transformation method using scutellum tissue from mature seeds (Toki et al. 2006). Here, we applied and modified this method to increase the transformation efficiency and reproducibility by adding the endosperm removal step and changing media composition and Agrobacterium strain in rice cultivar Dongjin.
Despite the many recent developments in the world of plant genetic manipulation, Agrobacterium-mediated transformation still remains a major method for transforming plant cells. Optimization of the conditions of tissue culture and co-cultivation is of critical importance. Several strains of Agrobacterium such as LBA4404, EHA101, EHA105, and GV3101 have been used for Agrobacterium-mediated transformation of rice (Rachmawati et al. 2004). LBA4404 is a derivative of the octopine-type strain Ach5, which contains the pTiAch5 plasmid (Jen and Chilton, 1986). EHA101 is a derivative of the succinamopine-type strain A281 containing pTiBO542, and EHA105 is a Km(S) derivative of EHA101 (Tsugawa et al. 2004). The efficiency of rice transformation is dependent on the cultivar and experimental conditions. In our experimental conditions, LBA4404 gave rise to higher efficiency transformation than did EHA105 (Figure 2). Agrobacterium strain EHA101 is known as a super-virulent strain and EHA105 is its derivative strain, so we had expected EHA105 to show higher transformation efficiency than the LBA4404 strain. The EHA105 strain harboring pCAMBIA1390sGFP (OD600 of approximately 0.1) was used previously for Agrobacterium infection of japonica rice cultivar ‘Nipponbare’ (Toki et al. 2006). However, when a high concentration of EHA105 cells (OD595 of approximately 0.1) was used for infection, and Agrobacterium cells were sometimes overgrown on 2N6-AS medium during co-culture under our experimental conditions with Oryza sativa cultivar Dongjin. Even though there was no critical different transformation efficiency depending on cell concentration used in LBA4404, we suggest that LBA4404 cells at OD595 0.01 are appropriate for stable and highly efficient transformation in Dongjin (Figure 2).
In angiosperm plants, endosperm, which is generally triploid, maintains an optimal physiological environment and supplies nutrition for the developing embryo. However, after embryogenic callus induction in rice tissue culture, nutrition for the growing calli is supplied through the medium. In addition, endosperm can be a source of contamination with overgrowth of Agrobacterium cells during tissue culture. Therefore, endosperm is not necessary or desirable after embryogenic callus induction. We found that transformation efficiency was increased after endosperm removal from scutellum-derived embryogenic calli compared to transformation efficiency in the presence of endosperm (Figure 3). When Agrobacterium infection without endosperm was performed, callus handling was also much easier.
We found out the optimal and reliable transformation conditions for Korean cultivar Dongjin using early infection of scutellum-derived calli with Agrobacterium. The high efficiency and reliability of this transformation method will contribute to make efficiency to identify the gene function of and to develop the useful transgenic rice.
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