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Delaying Seed Germination and Improving Seedling Fixation: Lessons Learned During Science and Payload Verification Tests for Advanced Plant EXperiments (APEX) 02-1 in Space

Jin Nakashima

Jin Nakashima

Plant Biology Division, The Samuel Roberts Noble FoundationArdmore, OK, United States
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Nakashima, Jin
, 
J. Alan Sparks

J. Alan Sparks

Plant Biology Division, The Samuel Roberts Noble FoundationArdmore, OK, United States
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Sparks, J. Alan
, 
John A. Carver

John A. Carver

Kennedy Space Center, Test and Operations Support ContractKennedy Space Center, FL, United States
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Carver, John A.
, 
Shawn D. Stephens

Shawn D. Stephens

Kennedy Space Center, Engineering Services ContractKennedy Space Center, FL, United States
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Stephens, Shawn D.
, 
Taegun Kwon

Taegun Kwon

Plant Biology Division, The Samuel Roberts Noble FoundationArdmore, OK, United States
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Kwon, Taegun
 und 
Elison B. Blancaflor

Elison B. Blancaflor

Plant Biology Division, The Samuel Roberts Noble FoundationArdmore, OK, United States
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Blancaflor, Elison B.
  
18. Jan. 2022
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Artikel-Kategorie: Research Article
Online veröffentlicht: 18. Jan. 2022
Seitenbereich: 54 - 67
DOI: https://doi.org/10.2478/gsr-2014-0005
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© 2014 Jin Nakashima et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1.

FR metal light box for delaying arabidopsis seed germination. (A) The light box consists of two parts. The lid, which contains the LED switch at the roof of the box, and a base where the Petri dish is positioned (arrows). (B) The ceiling of the metal lid showing the location of the FR LED, and the base showing placement of a Petri dish for FR light exposure. (C) Assembly of the metal box for exposing Petri dishes containing arabidopsis seeds to FR light. A 10-minute FR light treatment using this device, followed by maintaining plates in complete darkness, delayed seed germination until seeds were exposed to white light on ABRS.
FR metal light box for delaying arabidopsis seed germination. (A) The light box consists of two parts. The lid, which contains the LED switch at the roof of the box, and a base where the Petri dish is positioned (arrows). (B) The ceiling of the metal lid showing the location of the FR LED, and the base showing placement of a Petri dish for FR light exposure. (C) Assembly of the metal box for exposing Petri dishes containing arabidopsis seeds to FR light. A 10-minute FR light treatment using this device, followed by maintaining plates in complete darkness, delayed seed germination until seeds were exposed to white light on ABRS.

Figure 2.

ABRS and images of Petri dish on ABRS-GIS. (A) ABRS showing location of Petri dishes. Four out of six positions are indicated as yellow numbers (1,2,5,6). (B) Petri dish in position one of ABRS three days after planting (day 0 of installation on ABRS). Images of dishes were captured using the GIS unit (inset in A) of ABRS. Note that seeds have not yet germinated (arrows). (C) At two days after installation on ABRS, radicles can be seen protruding (arrows). (D) At seven days after installation on ABRS, seedlings have extended hypocotyls (arrows) and primary roots (arrowheads). Two genotypes (wild-type Col-0 and act2 act8 double mutant) were planted on the same plate during SVT and PVT. Note that at seven days after installation on ABRS, the act2 act8 double mutants, located on the right half of the plate (D), have primary roots with more wavy growth patterns.
ABRS and images of Petri dish on ABRS-GIS. (A) ABRS showing location of Petri dishes. Four out of six positions are indicated as yellow numbers (1,2,5,6). (B) Petri dish in position one of ABRS three days after planting (day 0 of installation on ABRS). Images of dishes were captured using the GIS unit (inset in A) of ABRS. Note that seeds have not yet germinated (arrows). (C) At two days after installation on ABRS, radicles can be seen protruding (arrows). (D) At seven days after installation on ABRS, seedlings have extended hypocotyls (arrows) and primary roots (arrowheads). Two genotypes (wild-type Col-0 and act2 act8 double mutant) were planted on the same plate during SVT and PVT. Note that at seven days after installation on ABRS, the act2 act8 double mutants, located on the right half of the plate (D), have primary roots with more wavy growth patterns.

Figure 3.

Quality of fixation of arabidopsis roots during SVT. (A, B) Semi-thin sections stained with Toluidine Blue O. Note that some root cells in the transition zone were plasmolyzed (arrows). (C, D) Roots labeled with monoclonal antibodies against fucosylated xyloglucan (CCRC-M1). Note the grainy fluorescence patterns and occurrence of punctate structures (arrows) in panels C and D. (E, F) TEM micrographs from the root transition zone. Note the extensive number of vacuoles (arrows) and irregularly shaped nuclei (arrowheads). Each set of micrographs are representative of four individual roots.
Quality of fixation of arabidopsis roots during SVT. (A, B) Semi-thin sections stained with Toluidine Blue O. Note that some root cells in the transition zone were plasmolyzed (arrows). (C, D) Roots labeled with monoclonal antibodies against fucosylated xyloglucan (CCRC-M1). Note the grainy fluorescence patterns and occurrence of punctate structures (arrows) in panels C and D. (E, F) TEM micrographs from the root transition zone. Note the extensive number of vacuoles (arrows) and irregularly shaped nuclei (arrowheads). Each set of micrographs are representative of four individual roots.

Figure 4.

KFTs preloaded with fixative used for in-orbit chemical fixation and containment of biological samples. (A) KFTs before actuation. The volume of fixative (red lines) is shown relative to the volume of air (yellow). (B) After actuation, the volume of air in contact with fixative increased. This could contribute to oxidation of the fixative. If an extra O-ring is added on the KFTs at the position indicated by the asterisk in panel B, the volume of air which potentially contacts fixative after the actuation would be reduced. This may lead to improvement in the quality of fixation for future microscopy work involving KFTs.
KFTs preloaded with fixative used for in-orbit chemical fixation and containment of biological samples. (A) KFTs before actuation. The volume of fixative (red lines) is shown relative to the volume of air (yellow). (B) After actuation, the volume of air in contact with fixative increased. This could contribute to oxidation of the fixative. If an extra O-ring is added on the KFTs at the position indicated by the asterisk in panel B, the volume of air which potentially contacts fixative after the actuation would be reduced. This may lead to improvement in the quality of fixation for future microscopy work involving KFTs.

Figure 5.

Adjustments made in composition and handling of fixative during PVT. Adjustments made included storing KFTs at 4° C and in the dark, both prior to fixation and after actuation (A); increasing glutaraldehyde concentration from 3% to 5% (B); and bubbling N2 gas over freshly prepared glutaraldehyde solution (C).
Adjustments made in composition and handling of fixative during PVT. Adjustments made included storing KFTs at 4° C and in the dark, both prior to fixation and after actuation (A); increasing glutaraldehyde concentration from 3% to 5% (B); and bubbling N2 gas over freshly prepared glutaraldehyde solution (C).

Figure 6.

Quality of seedling fixation during PVT. Serial semi-thin sections (0.25 μm in thickness) taken from a median longitudinal section of an arabidopsis primary root from seedlings grown in ABRS hardware and fixed in KFTs were stained with Toluidine Blue O (A and C) and processed by indirect immunofluorescence using a monoclonal antibody against fucosylated xyloglucan (CCRC-M1) (B and D). Note vacuolation (arrows in A) and plasmolysis (arrows in C) in Toluidine-Blue-O-stained roots. Each set of micrographs are representative of three individual roots.
Quality of seedling fixation during PVT. Serial semi-thin sections (0.25 μm in thickness) taken from a median longitudinal section of an arabidopsis primary root from seedlings grown in ABRS hardware and fixed in KFTs were stained with Toluidine Blue O (A and C) and processed by indirect immunofluorescence using a monoclonal antibody against fucosylated xyloglucan (CCRC-M1) (B and D). Note vacuolation (arrows in A) and plasmolysis (arrows in C) in Toluidine-Blue-O-stained roots. Each set of micrographs are representative of three individual roots.

Figure 7.

Additional tests to optimize seedling fixation in aldehyde for microscopy studies. Serial semi-thin sections (0.25 μm in thickness) taken from a median longitudinal section of an arabidopsis primary root from seedlings fixed at the laboratory that mirrored the duration of fixation on KFTs during SVT and PVT. Fixation was conducted with 4% paraformaldehyde and 3% glutaraldehyde (A to C), and 4% glutaraldehyde (D to F). Sections were stained with Toluidine Blue O (A and D) and indirect immunofluorescence labeling with monoclonal antibody against fucosylated xyloglucan (CCRC-M1) (B and E), and ultra-thin sections were observed under the transmission electron microscope (C and F). For comparison, a similar set of seedlings was fixed in 4% paraformaldehyde and 2.5% glutaraldehyde, and processed using routine laboratory protocols (e.g., no extended fixation) (G to I). Despite the slight plasmolysis and vacuolation, fixation quality with paraformaldehyde in the fixative generally improved. Each set of micrographs are representative of three individual roots.
Additional tests to optimize seedling fixation in aldehyde for microscopy studies. Serial semi-thin sections (0.25 μm in thickness) taken from a median longitudinal section of an arabidopsis primary root from seedlings fixed at the laboratory that mirrored the duration of fixation on KFTs during SVT and PVT. Fixation was conducted with 4% paraformaldehyde and 3% glutaraldehyde (A to C), and 4% glutaraldehyde (D to F). Sections were stained with Toluidine Blue O (A and D) and indirect immunofluorescence labeling with monoclonal antibody against fucosylated xyloglucan (CCRC-M1) (B and E), and ultra-thin sections were observed under the transmission electron microscope (C and F). For comparison, a similar set of seedlings was fixed in 4% paraformaldehyde and 2.5% glutaraldehyde, and processed using routine laboratory protocols (e.g., no extended fixation) (G to I). Despite the slight plasmolysis and vacuolation, fixation quality with paraformaldehyde in the fixative generally improved. Each set of micrographs are representative of three individual roots.

Impact of FR light treatment on germination and viability of four Arabidopsis ecotypes (Col-0, C24, Ler, and Ws)_ Percent germination and viability were obtained from a total of 30 seeds planted per ecotype and treatment regime_

2 Weeks
Non-treated Far-Red Light Treatment
Room Temperature 4°C Room Temperature 4°C
Ecotype Germination (%)* Viability (%)** Germination (%)* Viability (%)** Germination (%)* Viability (%)** Germination (%)* Viability (%)**
Col-0 100 100 76.7 100 0 100 3.3 100
C24 23.3 86.7 90 93.3 6.7 96.7 86.7 100
Ler 63.3 96.7 23.3 96.7 0 96.7 16.7 100
Ws 0 86.7 10 100 3.3 86.7 6.7 100
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