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Approaches for Surveying Cosmic Radiation Damage in Large Populations of Arabidopsis thaliana Seeds – Antarctic Balloons and Particle Beams

Brandon Califar
Brandon Califar
, 
Rachel Tucker
Rachel Tucker
, 
Juliana Cromie
Juliana Cromie
, 
Natasha Sng
Natasha Sng
, 
R. Austin Schmitz
R. Austin Schmitz
, 
Jordan A. Callaham
Jordan A. Callaham
, 
Bradley Barbazuk
Bradley Barbazuk
, 
Anna-Lisa Paul
Anna-Lisa Paul
 and 
Robert J. Ferl
Robert J. Ferl
   | Jul 21, 2020
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Article Category: Research Article
Published Online: Jul 21, 2020
Page range: 54 - 73
DOI: https://doi.org/10.2478/gsr-2018-0010
Keywords
© 2018 Brandon Califar et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1

Hardware and payload design for Brookhaven National Laboratory (BNL) exposure and Antarctica (ANT) high-altitude balloon flight. (A) A representative image of a 24 x 24 bin seed tray flanked by CR-39 Solid-State Nuclear Track Detector (SSNTD) sheets. The seed trays are modified 1536-well plates. Magnified views of the seed trays are shown below, including a close view of two individual bins. A single bin contains approximately 250 Arabidopsis thaliana seeds. (B) The custom 3D-printed holder used in the Antarctica payload and the biological payload it contained. (C) The modified T-25 flask containing the biological payload for irradiation at the NASA Space Radiation Lab (NSRL), located at the Brookhaven National Laboratory. (D) A diagram illustrating the individual components and configuration of the entire ANT payload for the high-altitude balloon flight in Antarctica. (E) The ANT payload (indicated by a red arrow) integrated onto the Boron And Carbon Cosmic Rays in the Upper Stratosphere (BACCUS) support structure. (F) Inflation of the balloon on the ice. (G) The path of the BACCUS payload over Antarctica, where blue indicates earlier time points and red indicates time points at the end of the flight. [Photos in E and R courtesy of Scott Miller – NASA CSBF]
Hardware and payload design for Brookhaven National Laboratory (BNL) exposure and Antarctica (ANT) high-altitude balloon flight. (A) A representative image of a 24 x 24 bin seed tray flanked by CR-39 Solid-State Nuclear Track Detector (SSNTD) sheets. The seed trays are modified 1536-well plates. Magnified views of the seed trays are shown below, including a close view of two individual bins. A single bin contains approximately 250 Arabidopsis thaliana seeds. (B) The custom 3D-printed holder used in the Antarctica payload and the biological payload it contained. (C) The modified T-25 flask containing the biological payload for irradiation at the NASA Space Radiation Lab (NSRL), located at the Brookhaven National Laboratory. (D) A diagram illustrating the individual components and configuration of the entire ANT payload for the high-altitude balloon flight in Antarctica. (E) The ANT payload (indicated by a red arrow) integrated onto the Boron And Carbon Cosmic Rays in the Upper Stratosphere (BACCUS) support structure. (F) Inflation of the balloon on the ice. (G) The path of the BACCUS payload over Antarctica, where blue indicates earlier time points and red indicates time points at the end of the flight. [Photos in E and R courtesy of Scott Miller – NASA CSBF]

Figure 2

Etching and examination of CR-39 SSNTDs to assess impact locations. (A) A representative CR-39 SSNTD exposed to intense mixed-beam radiation at BNL and etched showed densely distributed and unidirectional particle tracks. Scale bar = 1 mm. (B) A representative ANT CR-39 SSNTD exposed to cosmic radiation in the upper stratosphere showed sparse entry and exit cones at varying angles. Scale bar = 1 mm. (C) Conical holes in both the upper and lower planes of the etched CR-39 SSNTDs, viewed under higher magnification, illustrate the tracks that are indicative of a particle's passage through the sheet. Scale bars = 25 μm. (D) An image resulting from the X-ray Micro-Computed Tomography (Micro-CT) scan of a CR-39 SSNTD, illustrating how the paths of ions through the sheet can be mapped with high resolution.
Etching and examination of CR-39 SSNTDs to assess impact locations. (A) A representative CR-39 SSNTD exposed to intense mixed-beam radiation at BNL and etched showed densely distributed and unidirectional particle tracks. Scale bar = 1 mm. (B) A representative ANT CR-39 SSNTD exposed to cosmic radiation in the upper stratosphere showed sparse entry and exit cones at varying angles. Scale bar = 1 mm. (C) Conical holes in both the upper and lower planes of the etched CR-39 SSNTDs, viewed under higher magnification, illustrate the tracks that are indicative of a particle's passage through the sheet. Scale bars = 25 μm. (D) An image resulting from the X-ray Micro-Computed Tomography (Micro-CT) scan of a CR-39 SSNTD, illustrating how the paths of ions through the sheet can be mapped with high resolution.

Figure 3

Seed processing and screening procedures. (A) M0 seeds were de-integrated from specific bins in the seed tray(s) and organized into individual microcentrifuge tubes. (B) De-integrated seeds were wet sterilized with 70% ethanol and 50% bleach. (C) Once sterilized, seeds were planted on gridded Petri plates containing 0.5% Phytagel™ nutrient media. (D) After 14 days of growth, M0 plants were scored for germination and phenotypic traits; red-outlined grids highlight non-germinated seeds. (E) Atypical plants were imaged and tracked for further observation. (F) Atypical plants that displayed severely inhibited growth and development were frozen with liquid nitrogen.
Seed processing and screening procedures. (A) M0 seeds were de-integrated from specific bins in the seed tray(s) and organized into individual microcentrifuge tubes. (B) De-integrated seeds were wet sterilized with 70% ethanol and 50% bleach. (C) Once sterilized, seeds were planted on gridded Petri plates containing 0.5% Phytagel™ nutrient media. (D) After 14 days of growth, M0 plants were scored for germination and phenotypic traits; red-outlined grids highlight non-germinated seeds. (E) Atypical plants were imaged and tracked for further observation. (F) Atypical plants that displayed severely inhibited growth and development were frozen with liquid nitrogen.

Figure 4

Plants exhibiting mutant phenotypes selected for PacBio sequencing. Plants with a sufficient amount of fresh tissue weight to provide 1 μg of DNA for library generation (A–D) were sequenced individually. (A) Plant M0 ANT W3-2 D7 and (B) M0 ANT H16-2 K9 both exhibited development without growth of a primary root. (C) M1 BNL H13-1 H10 had delayed root growth by 14 days. (D) M1 BNL P18-3 F3 had only a single cotyledon, which then showed malformation. Plants from (E–J) were pooled due to the small amount of DNA extracted from each sample. (E) M0 ANT P19-1 H2 developed a single cotyledon with no other shoot or root structures, whereas (F) M0 ANT R2-1 E12 grew only a primary root. (G) M0 ANT P19-1 H3 cotyledons emerged without root growth. (H) M0 ANT R2-2 F7 and (I) M0 ANT P19-1 G12 both had cotyledons and a primary root only. (J) M0 ANT C18-2 C10 exhibited a fused cotyledon morphological mutation. All images are of plants aged 14 days old, with the exception of (B) for which only a 16-day-old image was available. Scale bars = 2 mm.
Plants exhibiting mutant phenotypes selected for PacBio sequencing. Plants with a sufficient amount of fresh tissue weight to provide 1 μg of DNA for library generation (A–D) were sequenced individually. (A) Plant M0 ANT W3-2 D7 and (B) M0 ANT H16-2 K9 both exhibited development without growth of a primary root. (C) M1 BNL H13-1 H10 had delayed root growth by 14 days. (D) M1 BNL P18-3 F3 had only a single cotyledon, which then showed malformation. Plants from (E–J) were pooled due to the small amount of DNA extracted from each sample. (E) M0 ANT P19-1 H2 developed a single cotyledon with no other shoot or root structures, whereas (F) M0 ANT R2-1 E12 grew only a primary root. (G) M0 ANT P19-1 H3 cotyledons emerged without root growth. (H) M0 ANT R2-2 F7 and (I) M0 ANT P19-1 G12 both had cotyledons and a primary root only. (J) M0 ANT C18-2 C10 exhibited a fused cotyledon morphological mutation. All images are of plants aged 14 days old, with the exception of (B) for which only a 16-day-old image was available. Scale bars = 2 mm.

Figure 5

Germination and mutation rates in irradiated seeds and plants. A. thaliana seeds irradiated at BNL and ANT exhibited significantly different germination and mutation rates from a non-irradiated control group at 14 days post-planting. (A) M0 Seeds exposed to heavy ions from a laboratory particle accelerator at BNL had a mean germination rate of 76%, whereas naturally occurring stratospheric radiation (ANT) resulted in a mean germination rate of 82%. The non-irradiated control had a mean germination rate of 98%. These results were statistically significant when compared to a control group. (B) Data for germination rates were also collected for the next generation (M1) of irradiated BNL seeds. Germination rates for the M1 BNL seeds were similar to the control group (at 98%) and significantly higher than the M0 BNL seeds. (C) Mutation rate was calculated based on the number of phenotypic aberrations observed per 100 germinated seeds. The mean mutation rates for the M0 of BNL and ANT were 12 and 4 mutations per 100 germinated seeds, respectively. In the control, the mean mutation rate was significantly lower at 1 per 100 seeds. (D) The BNL M1 showed a mean rate of 6 mutations per 100 seeds, which was significantly higher than the control, but significantly lower than the BNL M0 plants. Significance was determined via a Student's t-test and is denoted by asterisks (* = p<0.05 and ** = p<0.01).
Germination and mutation rates in irradiated seeds and plants. A. thaliana seeds irradiated at BNL and ANT exhibited significantly different germination and mutation rates from a non-irradiated control group at 14 days post-planting. (A) M0 Seeds exposed to heavy ions from a laboratory particle accelerator at BNL had a mean germination rate of 76%, whereas naturally occurring stratospheric radiation (ANT) resulted in a mean germination rate of 82%. The non-irradiated control had a mean germination rate of 98%. These results were statistically significant when compared to a control group. (B) Data for germination rates were also collected for the next generation (M1) of irradiated BNL seeds. Germination rates for the M1 BNL seeds were similar to the control group (at 98%) and significantly higher than the M0 BNL seeds. (C) Mutation rate was calculated based on the number of phenotypic aberrations observed per 100 germinated seeds. The mean mutation rates for the M0 of BNL and ANT were 12 and 4 mutations per 100 germinated seeds, respectively. In the control, the mean mutation rate was significantly lower at 1 per 100 seeds. (D) The BNL M1 showed a mean rate of 6 mutations per 100 seeds, which was significantly higher than the control, but significantly lower than the BNL M0 plants. Significance was determined via a Student's t-test and is denoted by asterisks (* = p<0.05 and ** = p<0.01).

Figure 6

Genomic rearrangements detected in sequenced ANT and BNL mutants. Translocations detected in the sequenced mutants from the (A) ANT M0 individual mutants, (B) ANT M0 Pooled Mutants, (C) BNL M1 individual mutants, and (D) Col-0 control datasets. Plots were generated in Circos from the filtered SV dataset for each sample. Translocation breakpoints detected by Sniffles are connected by links, which are color-coded according to the mutant in which the translocation was detected. Numeric labels on the periphery of the plots correspond to the distance (in Mbp) to that locus from the beginning of that chromosome's reference sequence. The number of translocations detected in each sample (post-filtering) is indicated next to its name.
Genomic rearrangements detected in sequenced ANT and BNL mutants. Translocations detected in the sequenced mutants from the (A) ANT M0 individual mutants, (B) ANT M0 Pooled Mutants, (C) BNL M1 individual mutants, and (D) Col-0 control datasets. Plots were generated in Circos from the filtered SV dataset for each sample. Translocation breakpoints detected by Sniffles are connected by links, which are color-coded according to the mutant in which the translocation was detected. Numeric labels on the periphery of the plots correspond to the distance (in Mbp) to that locus from the beginning of that chromosome's reference sequence. The number of translocations detected in each sample (post-filtering) is indicated next to its name.

Quantification of gDNA extractions via Qubit.

SampleFresh weight (mg)Qubit Conc. (ng/μl)Volume (μl)DINSample mass (ng)
Mutant1-M0ANT W3-2-D71024.2286.6678
Mutant2-M0ANT H16-2-K91042.0286.41176
Mutant4-M1BNL H13-1-H101025.6306.6768
Mutant5-M1BNL P18-3-F32049.4306.81482
Mutant7-M0ANT (Pooled)1515.7535.9833

Profile of the mixed-beam for cosmic radiation simulation used in the Brookhaven National Laboratory (BNL) seed treatment.

IonsEnergy (MeV)Dose (cGy)Dose rate
H25010.600.166
He2503.1700.166
O3501.0800.166
Ti3000.7350.166

Structural variants detected in the nuclear genomes of sequenced mutants.

SampleDELDUPINSINVINVDUP
Mutant1-M0ANT W3-2-D765533
Mutant2-M0ANT H16-2-K911222
Mutant4-M1BNL H13-1-H10541274
Mutant5-M1BNL P18-3-F383722
Mutant7-M0ANT (Pooled)325596
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