Optimization of regeneration and Agrobacterium tumefaciens-mediated transient transformation systems for Australian native extremophile, Tripogon loliiformis

2.1 Plant materials

Mature grains of Tripogon loliiformis, used for callus induction in this research, were collected from the Centre for Agriculture and Bioeconomy (CAB), QUT. The 5 weeks old fresh calli, initiated from the grains, were employed for regeneration and transformation experiments. The friable callus was nodular in shape, healthy and cream white in colour.

2.2 Media and chemicals

Different media and chemicals used are given in Supporting Tables 1 and 2.

2.3 Reporter genes and vector backbones

The reporter gene UidA from pCAMBIA2301 (Supporting Fig. 1), pENTR/D-TOPO® entry vector (Supporting Fig. 2) and pCE104-EYFP plant expression vector (Supporting Fig. 3) were collected from the CAB, QUT.

2.4 Seed sterilization and callus induction

Dehusked mature grains of T. loliiformis were washed thrice with sterile MilliQ water followed by surface sterilization with 70% ethanol for 30s with vortexing and re-washing for 3 times with MilliQ water to get rid of traces of ethanol. The grains were then soaked in 6.5% sodium hypochlorite (w/v) for 5min and vortexed 3 times at 1min interval followed by washing with MilliQ water for 7 times. The clean grains were subjected for imbibation in 0.05% agarose for 2h at room temperature. The pre-sterilized 20 aseptic grains were inoculated on petri dishes (90 × 15mm size) containing suitable callus induction media with appropriate concentrations of 2, 4-D semi-solidified by using 2.5gL⁻¹ of Gelzan.

2.5 Plant regeneration

After incubating the inoculated petri dishes at 27°C under dark condition inside growth cabinet for 5 weeks, the friable calli were selected and further multiplied by subculturing to the similar induction medium under dark for 3 weeks. These 8 weeks old calli clumps were then subcultured to the regeneration media for regenerating shoots and roots. 10 calli clumps were subcultured in each petri dish (90 × 14mm) containing 40mL of MS media supplemented with 30gL⁻¹ sucrose, and 2.5gL⁻¹ of solidifying agent Gelzan at pH 5.8 in addition to varying concentrations of BAP (0mgL⁻¹, 1.0mgL⁻¹; 2.0mgL⁻¹ and 3.0mgL⁻¹) and NAA (0mgL⁻¹; 0.25mgL⁻¹ and 0.5mgL⁻¹). All the plates were incubated at 27°C under dark inside growth cabinet for a week. Finally, the calli were exposed to light at 70µmoless⁻¹m⁻² white fluorescent light inside growth cabinet with a 16h light/ 8h dark cycle.

2.6 Preparation of constructs for Agrobacterium mediated transformation

The construct pCE104-UidA-EYFP was prepared by using TOPO cloning and Gateway LR cloning system. The amplification of UidA gene from the pCAMBIA2301 vector was done by PCR reactions employing Q5 High Fidelity DNA polymerase kit. After gel electrophoresis, the amplified gene was purified from agarose gel using a Freeze ‘N Squeeze DNA gel extraction spin column (Bio-Rad). The purified PCR product was cloned into pENTR/D-TOPO® vector and therefore the resulting pENTR-UidA (Supporting Fig. 4) was transformed into electro-competent cells of E. coli by electroporation. The pENTR-UidA plasmid purification was done using Wizard Plus SV Minipreps DNA purification system (Promega) for Sanger sequencing using Big Dye Terminator Cycle Sequencing Kit TM v3.1 (Applied Biosystems) and Vector NTI Advance 11 software was applied to analyse the sequencing data.

The digestion of the pENTR-UidA was executed using Cutsmart buffer and PvuI restriction enzyme. UidA gene together with the attL1 and attL2 recombination sites was transferred from pENTR vector into pCE104 destination vector using the Gateway™ LR Clonase™ II Enzyme Mix (Invitrogen). The LR reaction mixture was used to transform 50µL of thawed E. coli XL1 Blue chemically-competent cells following the heat shock method described by Inoue et al. (1990) and the construct pCE104-UidA-EYFP was subjected to Sanger sequencing for confirmation after analysing throughVector NTI Advance 11 software. The ultimate cloned product of pCE104-UidA-EYFP (Supporting Fig. 5) was transformed into Agrobacterium strains AGL1 and LBA4404 by electroporation. The AGL1 colonies with construct pCAMBIA2301 were collected from CTCB lab. The colonies were grown in 50mL LB liquid media with 25mgL⁻¹ rifampicin and 50mgL⁻¹ kanamycin and incubated 48h at 28°C for subsequent use for transformation. The primers used in every step of cloning are enlisted in Supplementary Table 3.

2.7 Agrobacterium-mediated transformation and GUS expression analysis

Fresh 5 week-old Tripogon calli were transformed following Agrobacterium-mediated transformation method for rice described in Hoang (2014) (Supporting Fig. 6) and for Brachypodium distachyon as described by Alves and colleagues (Alves et al., 2009) (Supporting Fig. 7) with 2 Agrobacterium strains, AGL1 or LBA4404 harboring single appropriate construct- pCE104-UidA-EYFP or pCAMBIA2301, were used for transformation. A 100µL aliquot of fresh Agrobacterium culture or 200µL of bacterial glycerol stock was added into 10mL liquid LB media (Supporting Table 1) with 50mgL⁻¹ kanamycin and 25mgL⁻¹ rifampicin in a 50mL falcon tube and shaken on a shaking incubator for 72h at 28°C, 200rpm. After that, PCR reactions and agarose gel electrophoresis were performed to verify the presence of appropriate construct within the culture.

Agrobacterium mediated transformation method described in Hoang (2014) was employed for the transformation of 5 weeks-old Tripogon calli. 40mL of ATM2 media (Supporting Table 1) supplemented with 50mgL⁻¹ kanamycin was mixed with 10mL growing Agrobacterium culture in liquid LB media in an autoclaved 250mL conical flask and kept shaking overnight at 28°C, 200rpm. On the day of transformation, following the transfer of each 25mL of Agrobacterium culture to a 50mL falcon tube, centrifugation of the cultures was done at 25°C, 3000rpm for 10min. The supernatant was discarded and the pellets were suspended in BRM media (Supporting Table 1) supplemented with 1µLmL⁻¹ of 200μM acetosyringone. For facilitating vir gene induction of Agrobacterium, the culture was then incubated at room temperature at a speed of 100rpm in a shaking incubator for 5h. Following a 5h incubation, an aliquot of 1mL from each falcon tube was taken to quantify the optical density (OD) employing a spectrophotometer at a wavelength of 600nm (OD₆₀₀) and the culture was centrifuged at 25°C, 3000rpm for 10min. The supernatant was discarded followed by suspending the pellets by vortexing in an appropriate amount of ATM4 media (Supporting Table 1) supplemented with 1µLmL⁻¹ of 200μM acetosyringone to acquire the OD₆₀₀ of 0.7. Simultaneously, 10mL of the preheated (45°C) ATM6 media (Supporting Table 1) was added to 20 calli clumps in a 50mL falcon tube. The calli were allowed to have heat shock by placing the falcon tube in water bath at 45°C for 5min followed by keeping in a 4°C fridge for 30min. After the cold shock, ATM6 media was decanted and 10mL of induced Agrobacterium culture was added. Before being centrifuged at 25°C, 1000rpm for 10min, the mixture was then shaken at 70rpm at room temperature for 5min. Following that, the calli in the Agrobacterium suspension left standing inside laminar hood at room temperature for 30min. The media was then discarded and the calli blotted dry on sterile Whatman filter paper. However, mock transformation was applied by placing some calli in ATM4 media without Agrobacterium. The inoculated calli were placed on co-cultivation media (Supporting Table 1) for 3 days at 28°C in dark growth cabinet.

Tripogon calli were also transformed using Agrobacterium-mediated transformation method for Brachypodium distachyon as described by Alves and colleagues (Alves et al., 2009) when 5 weeks-old. Before the day of transformation, 10mL liquid LB media with Agrobacterium strain harboring the genes of interest was transferred to a 250mL sterile conical flask and cultured overnight in 90mL MSB media (Supporting Table 1) supplemented with 45mgL⁻¹ of acetosyringone and 100mgL⁻¹ kanamycin at 28°C with a speed of 200rpm in a rotary shaker. On the following day, the overnight culture was transferred to 50mL falcon tube with 25mL in each tube and was centrifuged at 25°C, 3000rpm for 10min. The supernatant was discarded and the pellets in each falcon tube were suspended by vortexing in 25mL of MSB media supplemented with 45mgL⁻¹ of acetosyringone. The culture was kept in a shaker incubator at room temperature for 45min to urge the acceptable density. The optical density of 1mL of culture from each falcon tube was measured by spectrophotometer at a wavelength of 600nm (OD₆₀₀). The falcon tubes with culture were centrifuged at 3000rpm for 10min and the supernatant was decanted. The pellet was vortexed again in appropriate amount of MSB media supplemented with 45mgL⁻¹ of acetosyringone to prepare the OD₆₀₀ of the Agrobacterium suspension to 1.0. Approximately 20 Tripogon calli pieces were transferred into each 90 × 15mm petri plate and flooded with 15mL of Agrobacterium culture (OD₆₀₀ of 1.0) for 5min at room temperature. After inoculation, the calli were desiccated on sterile Whatman filter paper for 7min in laminar flow and then co-cultured on MSB3 media (Supporting Table 1) supplemented with 60mgL⁻¹ of acetosyringone for 3 days. Beside this, some calli were subjected to be mock-transformed in MSB media without any Agrobacterium strain.

Transient expression of UidA gene was analysed after three days of transformation following the strategy described by Jefferson et al. (1987). The calli were observed under a Zeiss Steri-2000-C stereomicroscope and pictures were captured with a digital microscope camera Progress®C5 (Jenoptic, Germany).

2.8 Data analysis

All the data were analysed by using the one-way ANOVA at P ≤ 0.05 in Minitab statistical software version 19.1.1. Tukey’s HSD tests were used to analyse the significant differences among the data. Data within the same parameter category followed by different letters are significantly different at P ≤ 0.05.

3.1 Establishment of regeneration system for Tripogon loliiformis

3.1.1

3.1.1 Friable callus induction of Tripogon lolliformis from mature grains

We chose to investigate the suitable concentration of 2,4-D for embryogenic callus induction from mature grains of Tripogon among the 4 concentrations (0.5, 1.0, 2.0 and 3.0mgL⁻¹) in MS media supplemented with 30gL⁻¹. The embryogenic callus was friable, globular in shape and cream white in colour (Fig. 1a). We observed that embryogenic callus induced in 1.0mgL⁻¹ 2,4-D after 5 weeks was the highest in percentage (73%) significantly compared to other concentrations (Fig. 1b). However, at 2.0mgL⁻¹ 2,4-D concentration, the percentage of callus induction (defined as percentage of seeds developing at least 1 callus) was not statistically different with 1.0mgL⁻¹ 2,4-D. Not enough amount of calli formed at low concentration (0.5mgL⁻¹) and high concentration (3.0mgL⁻¹) to proceed with further experiments of regeneration. As 2,4-D is a strong growth regulator, We hypothesized that the higher concentrations (2.0 and 3.0mgL⁻¹) might inhibit plant regeneration from calli masses and considered 1.0mgL⁻¹ as the optimum concentration for callus induction in Tripogon.

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Fig. 1

The initiation of callus on MS media supplemented with different concentrations of 2,4-D. a) Morphological features of embryogenic callus of Tripogon loliiformis. b) Mean percentage of callus induced in MS media with different concentrations of 2,4D. n = 10 (corresponding to 10 independent biological replications with 20 seeds each). Error bars represent standard error. Scale bar = 1 mm.

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3.1.2

3.1.2 Effect of different media in combination with optimised 2,4-D for callus induction

We investigated whether MS medium supplemented with 1.0mg L⁻¹ 2,4-D is the optimum concentration for callus induction. For this, an experiment was conducted with another medium that was used successfully for rice callus induction/transformation namely 2N6 - a modification of N6 medium (Hiei and Komari, 2008) supplemented with 1.0 and 2.0mgL⁻¹ 2,4-D (optimum concentration for higher percentage of callus induction inferred from previous experiment). The grains produced 72% of embryogenic callus after 5 weeks in MS medium supplemented with 1.0mgL⁻¹ of 2,4-D which is significantly higher compared to the percentages of calli obtained in 2 N6 medium supplemented with 1.0 (56%) and 2.0mgL⁻¹ 2,4D (51%), respectively (Fig. 2). We theorized that MS medium with 1.0mgL⁻¹ 2,4-D was most suitable for callus induction than 2N6 medium.

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Fig. 2

Callus induction from Tripogon grains on MS and 2 N6 media. (a) Mean percentage of callus induction in MS and 2 N6 media, n = 10 (corresponding to 10 independent biological replications with 20 seeds each). Error bars represent standard error. (b and c) MS media with 1.0mgL⁻¹ 2,4-D on 0 day and after 5 weeks, (d and e) MS media with 2.0mgL⁻¹ 2,4-D on 0 day and after 5 weeks, (f and g) 2 N6 media with 1.0mgL⁻¹ 2,4-D on 0 day and after 5 weeks and (h and i) 2 N6 media with 2mgL⁻¹ 2,4-D on 0 day and 5 weeks, Scale bar = 1 mm.

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3.1.3

3.1.3 BAP and NAA for green point emergence and regenerating miniature plantlets of Tripogon

Green points emerge from the creamy white calli when exposed to light and later form healthy shoots. We subcultured the calli induced in optimised media to MS medium supplemented with shoot and root inducing growth regulators (BAP and NAA) under the effect of light. The percentage of the green points (defined as percentage of calli developing at least 1 green point scored by eye) was recorded after 14 days of exposure to light (Fig. 3a). Almost all the calli obtained green points (100%) in MS media supplemented with (1) 2.0mgL⁻¹ BAP and 0.5mgL⁻¹ NAA combinations (Fig. 3b); (2) 3.0mgL⁻¹ BAP and 0.25mgL⁻¹ NAA and (3) 3.0mgL⁻¹ BAP and 0.5mgL⁻¹ NAA combinations when transferred to light for 14 days. The callus in control MS media without BAP and NAA fail to form any green points at the same time point. Green points also emerged in calli clumps when kept in media supplemented with other combinations of BAP and NAA but in lower percentages. Calli with green points failed to regenerate shoots and eventually died when subcultured on MS control and on MS medium with their respective BAP and NAA combinations for 3 weeks interval. However, the initiation of shoots ensued on medium with 2.0mgL⁻¹ BAP and 0.5mgL⁻¹ NAA after 3 weeks (Fig. 3c). This result reveals that higher concentrations of growth regulators in subsequent subculturing of calli was detrimental for regeneration for sensitive plant like Tripogon. We subcultured the callus with initiated shoots, obtained from 2.0mgL⁻¹ BAP and 0.5mgL⁻¹ NAA, for another 3 weeks on media with reducing amount of NAA into half though the amount of BAP was the same. Shoots emerged from each callus clumps in MS medium supplemented with 2.0mgL⁻¹ BAP and 0.25mgL⁻¹ NAA (Fig. 3d). Rooting was obtained from the base of each shoot clumps by subculturing the regenerated shoots in MS medium supplemented with 2.0mgL⁻¹ BAP and 0.25mgL⁻¹ NAA within 3 weeks (Fig. 3e). The procedure for regenerating plantlets from embryogenic calli of Tripogon loliiformis is represented in Fig. 4.

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Fig. 3

Regeneration of Tripogon loliiformis plantlets from calli. a) Mean percentage of green points coming on MS medium containing different concentrations of BAP and NAA. n = 6 (corresponding to 6 independent biological replications with 10 calli each). Error bars represent standard error, (b) emergence of green points on calli after 2 weeks and (c) shoot initiation from calli after 5 weeks on MS medium with 2.0mgL⁻¹ BAP and 0.5mgL⁻¹ NAA, (d) shoot regeneration after 8 weeks and (e) profuse rooting from shoots after 11 weeks in MS medium supplemented with 2.0mgL⁻¹ BAP and 0.25mgL⁻¹ NAA. Calli were exposed in 16 h light/8h dark. Scale bar = 14 mm.

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Fig. 4

Tripogon loliiformis tissue culture procedure.

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3.2 Transient expression UidA gene after Agrobacterium-mediated transformation of T. loliiformis

Tripogon loliiformis is closely related to rice (Oryza sativa) as both are monocotyledonous plants belonging to the grass family. We conducted experiment for Tripogon transformation following the high efficiency rice transformation protocol by Hoang (2014) where embryogenic calli raised from rice grains were used as the explants. Histochemical GUS staining for transient UidA gene expression of 5 weeks old calli after 3 days post transformation showed intense blue colouration in 59% of the transformed calli. However, the control embryonic calli without Agrobacterium transformation did not turn blue indicating that gene has been successfully delivered to the embryogenic transformed calli after Agrobacterium infection (Fig. 5).

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Fig. 5

UidA gene expression assay after 3 days of transformation following rice transformation protocol (Hoang 2014). (a) Calli transformed with AGL1pCE104-UidA- EYFP, (b) non-transformed control calli, n = 3 (corresponding to 3 independent biological replications with 10 calli each) . Scale bar = 1 mm.

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We also followed Brachypodium distachyon transformation protocol developed by Alves et al. (2009) because Brachypodium is a monocot grass and embryogenic calli is the explant for transformation like Tripogon. This protocol with few modifications was experimented for transforming Tripogon calli by using two different Agrobacterium strains viz. AGL1 (pCE104-UidA-EYFP and pCAMBIA2301) and LBA4404 (pCE104-UidA-EYFP). 61% of calli expressed UidA gene for transformation performed with AGL1 (pCE104-UidA-EYFP), followed by 58% and 36% GUS expression for LBA4404 (pCE104-UidA-EYFP) and AGL1 (pCAMBIA2301), respectively in the transient expression essay of UidA gene at 3 days after transformation (Fig. 6).

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Fig. 6

UidA gene expression in Tripogon calli 3 days post-transformation with Agrobacterium strains AGL1 pCE104-UidAEYFP, AGL1 pCAMBIA2301 and LBA4404 pCE104-UidA-EYFP following Brachypodium transformation protocol. (a) AGL1 pCE104-UidA-EYFP, (b) AGL1 pCAMBIA2301, (c) LBA4404 pCE104-UidA-EYFP, (d) control, (e) Percentages of GUS stained calli. n = 3 (corresponding to 3 independent biological replications with 10 calli each). Scale bar = 1 mm.

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