Particle bombardment – mediated gene transfer and GFP transient expression in Seteria viridis

ABSTRACT

Setaria viridis is one of the most important model grasses in studying monocot plant biology. Transient gene expression study is a very important tool in plant biotechnology, functional genomics, and CRISPR-Cas9 genome editing technology via particle bombardment. In this study, a particle bombardment-mediated protocol was developed to introduce DNA into Setaria viridis in vitro leaf explants. In addition, physical and biological parameters, such as helium pressure, distance from stopping screen to the target tissues, DNA concentration, and number of bombardments, were tested and optimized. Optimum concentration of transient GFP expression was achieved using 1.5 ug plasmid DNA with 0.6 mm gold particles and 6 cm bombardment distance, using 1,100 psi. Doubling the bombardment instances provides the maximum number of foci of transient GFP expression. This simple protocol will be helpful for genomics studies in the S. viridis monocot model.

Introduction

Particle bombardment-mediated transformation is an alternative method that is suitable and efficient for many plant species. It facilitates DNA delivery into intact plant cells, through simultaneous multiple gene transfers with no biological constraints or host limitations.¹ Particle bombardment is also employed for DNA delivery in transient gene expression studies to investigate the plant gene expression, and for its ability to introduce DNA directly into different tissues.² Different plant organ tissues have been used as bombardment targets: pollen grains,³ immature embryos,⁴ embryogenic calluses,^5,6 embryogenic suspensions,⁷ hypocotyls,^8,9 epicotyls,^10,11 flowers,¹² fruits,^13–15 and root hairs.¹⁶ Recently, CRISPR-Cas9 genome editing and transformation has also used particle bombardment.¹⁷

S. viridis is a worldwide model monocot crop with the common name of “green foxtail.” It is a target of significant academic and industry research, and an important model plant in the study of monocot biology. S. viridis belongs to the Poaceae family, subfamily Panicoideae, which includes the most agronomically important grass crops. Besides being a crop, it is more closely related than B. distachyon to many of the most promising bioenergy grasses, including maize, sorghum, Miscanthus spp., switchgrass, and sugarcane. Additionally, Setaria italica was domesticated from S. viridis nearly 8,000 years ago.¹⁸

S. viridis has a number of characteristics that make it ideal as a model plant for genetics and functional genomics studies. Its small size (10–15 cm), short life cycle (6–9 weeks depending on photoperiod conditions), and prolific seed production (13,000 seeds/plant) are all traits desirable in genetic models.¹⁹ As whole-genome sequencing projects progress at an impressive rate^20,21 it is likely that new model organisms will emerge to address specific challenges in agriculture, such as biotic and abiotic stress tolerance. Agrobacterium-mediated transformation widely used to transform via seed-derived callus,^22,23 and spike dip.²⁴ This is the first report of transient GFP expression transformation of leaf explants in Setaria viridis using an alternative particle bombardment system.

Materials and methods

Plant materials and growth conditions

Setaria viridis mature seeds were treated with 20% bleach with 2–3 drops of Tween 20 for 20 min, and then the seeds were washed 3–5 times with sterile water for 10 min. Sterilized seeds were germinated in 100 × 20 mm petri dishes containing Murashige and Skoog (MS) medium²⁵ supplemented with 3% sucrose and 0.8% agar, at pH 5.6–5.8. Seeds were germinated under white florescent light at a photon flux of 24 in dark conditions for 5 days in a growth room with μmolm−2S−2 for 16/8-h light/dark photoperiod at 25°C ± 2°C. All media were sterilized at 120°C for 20 min.

Explant preparation and osmotic medium

Leaves were excised from 7–14-d-old seedlings and cut into 0.75 cm2 segments that included the midrib. Excised explants were placed adaxial side up in 25 × 100 mm petri dishes containing osmotic medium MS salts supplemented with sucrose vitamins for 4–5 hours.

Plasmid constructs

The plant transformation vector used in this study was PCAMBIA 1304 carries a selective marker gene, hygromycin phosphotransferase (hptII), conferring hygromycin resistance and a fusion between the reporter genes coding for glucuronidase (GUS, uidA) and a green fluorescent protein (GFP), both driven by a 35 S promoter from the cauliflower mosaic virus (CaMV). Plasmid DNA was transformed into E. coli DH5 cells. High-quality DNA for particle bombardment transformation was prepared with Qiagen Midiprep (Qiagen GmbH, Germany) according to the manufacturer's instructions. Supercoiled plasmid DNA was directly employed in transformation experiments or linearized by digestion with EcoR1 enzyme (New England Biolabs). The DNA was precipitated with two volumes of ethanol at room temperature by vortexing, and then air-dried at room temperature for 20 min. The DNA pellet was resuspended in 50 ul of TE buffer or water.

Particle bombardment

The plasmid DNA was precipitated onto gold particles according to the instruction manual for the Biolistic PDS-1000/He (BioRad) device. The gold particles were stored at 60 mg/ml in absolute alcohol, and the suspensions were vortexed vigorously for 3 minutes to get rid of aggregated lumps. This step was repeated 3 times; the suspensions were centrifuged for 1 minute at 10000 rpm, and the supernatant was discarded. The pellet was resuspended in 1 ml of sterile distilled water, vortexed, and centrifuged for 1 min, before the supernatant was discarded. This step was also repeated 3 times, and the resulting pellet was resuspended in 1 ml of sterile distilled water and 50 ul aliquoted (for 6 bombardments) each in microbes, while the vortexing of the suspension continued.

Using 1.5 ml Eppendorf tube, 1 ug plasmid DNA and 50 ul of CaCl2 (2.5 M) were added and vortexed gently. A further 20 ul of spermidine (0.1M) was added and mixed well; subsequently, this mixture was vortexed for 3 minutes then centrifuged for 30 s at 10000 rpm, and the supernatant was discarded. The pellet was washed with 250 ul of 100% alcohol, and the final pellet was resuspeded in 60 ul of 100% alcohol. 9 ul of aliquoted was then loaded onto the center of the macrocarrier, and given a few minutes to dry well before it was used for the bombardment. In this study, optimization of the physical parameter was carried out under the following conditions: rupture disc pressure (650, 1100, and 1350 psi), distance from stopping careen to the target tissue (3 and 6 cm), and number of bombardments (1 and 2 times) per plate.

Microscopy

GFP expression in particle bombardment in vitro leaves was monitored with the OLYMPUS stereomicroscope supplied with camera, GFP filter and ultraviolet illumination source set for excitation between 455 and 490 nm and emission wavelength of 525–550 nm.

Data analysis

A complete randomized design was used in all experiments, and analyses of variance and mean separations were performed using Duncan's Multiple Range Test (DMRT). Statistical significance was determined at 5% level.

Results and discussion

This study demonstrated that particle bombardment transformation has several advantages over Agrobacterium-mediated transformation. These include plasmid construction, tissue type, independence of species and genotype, and having easy protocols to follow.

Effect of helium pressure

The helium gas tank is one of the most important material for particle bombardment studies. Some plant tissues, like calluses, embryos, or leaves, vary in their thickness and surface characteristics; therefore, it is of vital importance to optimize the parameter needed for each tissue type individually.²⁶ High gas pressure can cause more damage to the target cells than low pressure; however, low gas pressure can lead to insufficient penetration of the target tissues by the gold micro-carrier. This study used 0.6µ gold particles under different pressures of 650, 1100, and 1350 psi. The 1100-psi helium pressure condition gave the highest transient GFP expression (Fig. 2A), compared to the 650 and 1350-psi conditions. Similar results have been reported.²⁷

Figure 2.

Effect of different parameters on GFP transient expression in S. viridis.

Effect of number of bombardments

Leaf explants were bombarded either once or twice per petri dish, in order to analyze the effect of the number of bombardments on the efficiently of GFP transient expression. A maximum of 80% transient GFP foci was achieved when using bombardment two times (Fig. 2C), while single bombardments achieved only 65% of the transient GFP foci. However, bombardment at two times per disc was associated with cell damage, and necrosis was observed.

Effect of osmotic treatment

In the current study, leaf explants underwent two different osmotic treatments: mannitol plus sorbitol, and mannitol with sucrose. They were treated 5 hours before bombardment and 18 hours post-bombardment, and then their GFP expression was evaluated. A maximum of 81% of the transient GFP foci was observed (data not shown) when using the sorbitol and mannitol combination.

Effect of distance

The distance between stopping screens and the target tissues also produced a recorded effect on the transient GFP foci expression and the efficiency of the transformation. This distance is necessary for allowing the even spread of the DNA microcarrier onto the target tissue without causing damage to the tissues.^28,27,29 A highest GFP expression average of 142 spots (Fig. 1D) was observed when a 6-cm distance (Fig. 2B) was used with an 1100-psi pressure. A shorter distance (3 cm) was observed to be associated with tissue damage and a decreased GFP foci level of the leaf surface. At a distance of 9 cm, the number of GFP positive spots was significantly reduced to 65 spots, due to the decreased velocity of the bombardment force, which led to fewer cells receiving DNA.

Figure 1.

Wild-type control and transient green fluorescent protein (GFP) expression of in vitro leaves. A. wild-type control leaf (bright light), B. wild type control leaf with GFP filter, C. bombarded in vitro leaf (bright light) D. the same leaf showing GFP transient expression with GFP filter.

Effect of DNA concentration

DNA precipitation with gold particles usually determines the potential amount of DNA delivered into the target tissues. In the present study, the three different ranges of plasmid DNA (0.5, 1.0, 1.5, 2.0 ug) were observed, and related data were collected. Among the three concentrations, 1.5 ug DNA gave the highest GFP spots expression in the leaf explants, and the low 0.5 ug concentration resulted in a lower number of spots per bombardment. However, the higher concentration of 2.0 ug or more DNA was observed to cause more particle aggregation and to target the leaf surfaces unevenly (data not shown) Similar observations were also reported.³⁰ Here, GFP activity in the surface of the bombarded in vitro leaf was observed after 24 or 48 hours bright light (Fig. 1C) GFP filter (Fig. 1D) vs. control bright light and GFP filter (Fig. 1A, B) respectively.

Conclusion

This is the first report using particle bombardment with in vitro leaf of transient GFP expression in S. viridis. Agrobacterium leaf infiltration is extremely difficult, due to the incredibly thin leaf structure and small area. This makes this alternate system very useful for monocot crops and improving the transgenic and genomic editing studies in the near future.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

MM thank the University Grants Commission (UGC-JRF), Government of India and The Mashav fellowship program, Israel.