Journal of Agricultural Technology 2014, Vol. 10 (2): 467-474红包怎么做灯笼
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The floral-dip method for rice (Oryza sativa ) transformation
Rod-in, W., Sujipuli, K. and Ratanasut, K.*
Department of Agricultural Science, Faculty of Agriculture, Natural Resources and Environment, Naresuan University, Phitsunulok, Thailand
Rod-in, W., Sujipuli, K. and Ratanasut, K. (2014). The floral-dip method for rice (Oryza sativa ) transformation. Journal of Agricultural Technology 10(2):467-474.
We developed an effective floral dip, Agrobacterium -mediated transformation method for rice (Oryza sativa L.) cultivar RD41. A. tumefaciens strain AGL1 harboring the binary vector pCAMBIA1304 carrying the gusA gene (AGL1-1304) was ud to infect rice spikelets via the floral-dip method. The tip-cut spikelets of the rice inflorescence stage 51 (beginning of panicle emergence: tip of inflorescence emerged from sheath) were dipped in the Agrobacterium AGL1-1304 suspension and co-cultivated at 25 °C for 3 d. The target sites of transformation were detected by histochemical GUS assay. Eighty thr
ee of six hundred and fifty two, 12.73%, of rice spikelets dipped were positive for GUS activity in various tissues. Tissues positive for GUS staining included female organs (ovary, stigma, style and lodicule), male organs (anther and filament), and lemma. The primary target of the floral-dip transformation in rice was the anther which had the highest transformation efficiency (89.16%) whereas ovary transformation was rare (7.23%). In addition, we demonstrated that most of the pollen in the GUS-stained anthers expresd GUS activity. This result indicated that the germ-line cells were transformed and the transgene can be expresd. Our results suggest that floral-dip transformation is a simple potential tool for production of transgenic rice with no requirement of tissue culture.
Keywords: Agrobacterium , Floral dip, GUS, Rice, Transformation
十四行诗莎士比亚Introduction
The first in planta flora-dip, Agrobacterium -mediated transformation, was successfully developed for Arabidopsis (Clough and Bent, 1998). This method is easy, convenient and economical and eliminates the microbial contamination which is commonly found in the tissue culture required for plant regeneration (Li et al., 2010). The disadvantages of the floral-dip method include low transform
ation efficiency, requirement of many flowers and eds, no specific target site of organ transformation, and limitations in the application of this method to all plant species. At prent, this method has been successfully applied to various plant species, e.g., Arabidopsis thaliana * Corresponding author: Ratanasut, K.; e-mail: kumropr@nu.ac.th
Journal of Agricultural Technology 2014 Vol. 10(2): 467-474
Available online ISSN 1686-9141
(Clough and Bent, 1998; Das and Joshi, 2011), Medicago truncatula (Trieu et al. 2000), Raphanus sativus (Curtis and Nam, 2001), Solanum lycopersicum (Yasmeen et al., 2009), Triticum aestivum (Zale et al., 2009), Brassica Napus (Li et al., 2010), Melilotus alba (Hirsch et al., 2010), Camelina sativa (Liu et al., 2012), and Zea Mays(Mu et al., 2012). However, there has not been reported on the floral-dip transformation in rice (Oryza sativa L.) yet.280年
Rice is one of the most versatile cereal crops and staple foods in the world, but its quality and yield have been decread from the destruction of dias and inct pests, and suboptimal environmental conditions. Genetic modification has been ud in rice for studying gene function and improving agricultural traits to gain resistance to both biotic and abiotic stress, for example, the im
provement of rice tolerant to drought and salt stress (Rohila et al., 2002; Zhang et al., 2010), the engineering of inct resistant rice (Ye et al., 2009), and the production of rice resistant to the sheath blight dia (Molla et al., 2013).
As tissue culture is labor intensive and slow, and frequently generates transformed plants harboring undesirable mutations from somaclonal variation (Bent, 2000; Clough and Bent, 1998), floral-dip transformation has no requirement of tissue culture and would be an advantageous method for rice improvement.
In this study, we evaluated the u of the floral-dip transformation method to introduce a model gene, gusA, driven by the CaMV35S promoter into rice. The primary goal of this study was to identify the sites of productive transformation in the floral-dip procedure for Agrobacterium-mediated transformation of rice. It was our hope to identify a potential tool for production of the transgenic rice via the floral-dip transformation method.
Materials and methods
Plant materials and growth conditions
香芹炒鸡蛋Rice (Oryza sativa L.) cultivar RD41 obtained from the Phitsanulok Rice Rearch Center, Thailand was ud in this study. The RD41 eds were soaked in water for 2 d and then placed in a moist filter cloth until germination. The germinated eds were planted in a pot containing fertile and wet clay. The 1-2 week-old edlings were moved to a new pot, three plants per pot. The rice plants were grown in the greenhou at Naresuan University, Phitsanulok province, Thailand.
The inflorescences at stage 51 (the beginning of panicle emergence: tip of inflorescence emerged from sheath) according to the BBCH-scale (Lancashire et al., 1991) were ud for the floral-dip transformation.
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Agrobacterium strain and plasmid
A. tumefaciens strain AGL1 harboring pCAMBIA1304 was ud for the floral-dip transformation. The binary vector pCAMBIA1304 contains the hygromycin phosphotransfera (hptII ) gene, and the reporter genes, green fluorescent protein (gfp ) and β-glucuronida (gusA ), under the control of the CaMV35S promoter (Figure 1).
已知悉Fig. 1. Schematic diagram of the pCAMBIA1304 T-DNA region. The hptII , gfp and gusA
genes are under the control of the CaMV35S promoter
Agrobacterium culture and inoculum
A. tumefaciens strain AGL1 harboring pCAMBIA1304 was grown in 5 ml of Luria broth (LB) medium supplemented with kanamycin (50 mg/l) and rifampicin (40 mg/l) at 28°C and 250 rpm for 2 d. One milliliter of the Agrobacterium suspension culture was inoculated in 250 ml of LB medium. The culture was grown at 28°C and 250 rpm until reaching the stationary pha OD 600 0.8–1.0. Bacterial cells were collected by centrifugation at 5600xg for 3 min. The cell pellets were resuspended in the inoculation medium which was modified from Clough and Bent (1998). The inocu
lation medium was compod of basal medium Murashige and Skoog (MS) medium (Murashige and Skoog, 1962), 5% (v/v) sucro, 44 nM benzylaminopurine, and 0.075% (v/v) of the surfactant Tween-20 (Das and Joshi, 2011). The pH of the inoculation medium was adjusted to 5.7 for dipping (Clough and Bent, 1998).
Floral dip transformation
Rice inflorescences at stage 51 as mentioned above were ud for transformation. The tips of lected rice spikelets were cut off before the inflorescence was dipped in the Agrobacterium inoculation medium. Dipping was carried out for 1 min. Each dipped inflorescence was covered with a plastic bag (to maintain humidity) at 25°C for 3 d.
GUS Assay
The achievement of transformation was evaluated by the GUS assay in the dipped spikelets according to the method of Jefferson et al. (1987). The dipped rice spikelets were immerd in GUS staining solution containing 50 mg/ml 5-bromo-4-chloro-3-indolyl-β-D glucuronida (X-gluc), 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, 1 M Na2PO4, 0.5 M EDTA (pH 7.0), 0.5% Triton X-100 and 20% Methanol. The GUS staining spikelets were incubated in the dark overnight at
37°C. The stained spikelets were immerd in methanol: acetic acid (3:1) and 70% ethanol overnight at room temperature to remove the chlorophyll. The transient GUS activity of spikelets was recorded as blue spots (irrespective of size) using a microscope and photographed.
Results and discussions
GUS expression in transformed tissues of rice flowers
The floral-dip transformation for rice was investigated bad on the floral-dip method for Arabidopsis(Clough and Bent, 1998) with some modifications as mentioned above. We examined the target tissues for the gusA transformation by the GUS assay.
Eighty-three of six hundred and fifty-two dipped spikelets (12.73%) showed GUS expression. GUS activity was detected in various tissues of the GUS-stained spikelets, i.e., lemma, male organs (anther and filament), and female organs (ovary, stigma, style and lodicule) (Figure 2). Among the tissues, the anther, a major target, was commonly transformed (89.16%) while transformation of the ovary, another important target, was rare (7.23%) (Table 1). To generate the transgenic plants, the target tissues of the floral-dip transformation are the xual organs, the ovary and anther which carry the germ-line cells, ovules and pollen, respectively. The results indicate that the anthers are the
primary site of the floral-dip transformation in rice. Our results differed from tho of previous rearch with floral-dip transformation in Arabidopsis which found that the female germ line was the primary site of transformation whereas the male germ-line transformation was rare (Bechtold et al., 2000; Desfeux et al., 2000; Ye et al., 2009).
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Fig. 2. Analysis of GUS activity in rice spikelets after floral dip transformation: a)-c). The dipped spikelet with no GUS expression in any tissues; d) the dipped spikelets with GUS activity in lemma (Le); e) the dipped spikelets with GUS activity in anthers (An) and filaments (Fi); and f) the dipped spikelets with GUS activity in lodicules (Lo), ovary (Ov), stigma and style (S). Bar = 1 mm.
Table 1. The efficiency of the floral-dip transformation in different tissues of rice spikelets. The data show target sites of transformation from eighty-three GUS-stained spikelets
Floral tissues
Number of GUS-stained spikelets Transformation efficiency (% ) Ovary
黄帝6 7.23 Stigma
5 6.02 Style
7 8.43 Lodicule
9 10.84 Anther
74 89.16 Filament 3 3.61
Determination of GUS expression in the pollens of the GUS-stained anthers
To confirm the male germ-line transformation, pollen from the GUS-stained anthers were examined for staining under a microscope. The result revealed that most of pollen obtained from the GUS-stained anthers showed GUS activity (Figure 3). The staining confirmed that the male germ line was the predominant target of the floral-dip transformation in rice. Our
result
472 suggests that the floral-dip transformation is a potential method to produce transgenic rice. However, determination of the viability and germination efficiency of transformed pollen is an esntial step in confirming the ufulness of floral-dip transformation in rice.
Fig. 3. GUS expression in pollens of the dipped spikelets: (a), pollens from the GUS-negative anthers; (b), pollens from the GUS-positive anthers. The photographs were taken under a compound microscope at 200x magnification. Blue-stained pollens indicate GUS expression. Bar = 100 µm.
七十二小时打一字Conclusion
In this study, we developed a floral-dip method for rice transformation by modifying the method ud for Arabidopsis (Clough and Bent, 1998). GUS assays of the rice spikelets dipped in the suspension of A. tumefaciens strain AGL1 harboring pCAMBIA1304 showed that 12.73% of the treated spikelets exhibited GUS activity in various floral tissues. The anther, a major target of the floral-dip transformation in rice, was transformed at a rate of 89.16%. In contrast, ovary transformation was rare. We also demonstrated that most of the pollen in the GUS-stained anthers showed GUS activity. Our results indicate that floral-dip transformation is a simple, potentially uful method for production of transgenic rice.
Acknowledgements
This work was supported by the rearch grant R2556B036 from Naresuan University, Thailand. We would like to thank Associate Professor Dr. Sompong Te-chato from Prince of Songkla University for
kindly providing A. tumefaciens strain AGL1 harboring pCAMBIA1304.
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