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Live imaging of developmental processes in a living meristem of Davidia involucrata (Nyssaceae).

Jerominek M, Bull-Hereñu K, Arndt M, Claßen-Bockhoff R - Front Plant Sci (2014)

Bottom Line: The growing meristem was observed for 30 days, the longest live observation of a meristem achieved to date.D. involucrata exemplarily shows that live-ELM gives new insights into developmental processes of plants.In addition to morphogenetic questions such as the transition from vegetative to reproductive meristems or the absolute timing of ontogenetic processes, this method may also help to quantify cellular growth processes in the context of molecular physiology and developmental genetics studies.

View Article: PubMed Central - PubMed

Affiliation: Institut für Spezielle Botanik, Johannes Gutenberg-Universität Mainz, Germany.

ABSTRACT
Morphogenesis in plants is usually reconstructed by scanning electron microscopy and histology of meristematic structures. These techniques are destructive and require many samples to obtain a consecutive series of states. Unfortunately, using this methodology the absolute timing of growth and complete relative initiation of organs remain obscure. To overcome this limitation, an in vivo observational method based on Epi-Illumination Light Microscopy (ELM) was developed and tested with a male inflorescence meristem (floral unit) of the handkerchief tree Davidia involucrata Baill. (Nyssaceae). We asked whether the most basal flowers of this floral unit arise in a basipetal sequence or, alternatively, are delayed in their development. The growing meristem was observed for 30 days, the longest live observation of a meristem achieved to date. The sequence of primordium initiation indicates a later initiation of the most basal flowers and not earlier or simultaneously as SEM images could suggest. D. involucrata exemplarily shows that live-ELM gives new insights into developmental processes of plants. In addition to morphogenetic questions such as the transition from vegetative to reproductive meristems or the absolute timing of ontogenetic processes, this method may also help to quantify cellular growth processes in the context of molecular physiology and developmental genetics studies.

No MeSH data available.


Twelve representative images from the time-lapse videos (Video S1, S2), showing the development of a male FU, from flower (A–D) and stamen initiation (EF) to anther development (G–K). Due to meristem growth the magnification was changed between (A–F) (Video S1) and (G–L) (Video S2). The corresponding bar lines are indicated in (A,G). The white and black arrows mark two individual primordia at different developmental states. The outer primordia (black arrow) are clearly initiated later than the inner ones (white arrow). Videos available at www.spinningspecies.com/davidia.html.
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Figure 3: Twelve representative images from the time-lapse videos (Video S1, S2), showing the development of a male FU, from flower (A–D) and stamen initiation (EF) to anther development (G–K). Due to meristem growth the magnification was changed between (A–F) (Video S1) and (G–L) (Video S2). The corresponding bar lines are indicated in (A,G). The white and black arrows mark two individual primordia at different developmental states. The outer primordia (black arrow) are clearly initiated later than the inner ones (white arrow). Videos available at www.spinningspecies.com/davidia.html.

Mentions: Since common binocular microscopes have angular optical paths that produce a shift of images in a stack, we used a monocular microscope (Leitz Wetzlar, Figure 2). Photographs were taken with a Canon Powershot G9 that was mounted on the eyepiece and triggered by a computer (Canon CameraWindow). For an automated stacking procedure a stepper motor was adapted to the focus wheel of the microscope and controlled by a four-channel motor driver circuit (L298N). For illumination a LED light was switched on during the image capture process. The whole stacking cycle was controlled by an open-source microcontroller (Arduino Leonardo) that was connected to the motor driver to change the focus. The camera was triggered by an implemented keyboard source code via a computer and the LED was controlled via a relay. Each cycle began with the activation of the LED and was followed by raising the microscope to the highest focus point. The next two steps that triggered the camera and moved the microscope to a lower focus position were repeated until the lowest focus point was reached (70 times). To finish the cycle the LED was turned off. For live imaging the cycle was triggered and repeated once per hour by a timer (remote control for cameras). Since the meristem grew, the focus range was checked twice a week and manually readjusted. After the meristem became too large the magnification was reduced from a 10-fold to a 4-fold objective (comp. Figures 3A–F and G–L). Accordingly, live observation is divided into two time lapse videos.


Live imaging of developmental processes in a living meristem of Davidia involucrata (Nyssaceae).

Jerominek M, Bull-Hereñu K, Arndt M, Claßen-Bockhoff R - Front Plant Sci (2014)

Twelve representative images from the time-lapse videos (Video S1, S2), showing the development of a male FU, from flower (A–D) and stamen initiation (EF) to anther development (G–K). Due to meristem growth the magnification was changed between (A–F) (Video S1) and (G–L) (Video S2). The corresponding bar lines are indicated in (A,G). The white and black arrows mark two individual primordia at different developmental states. The outer primordia (black arrow) are clearly initiated later than the inner ones (white arrow). Videos available at www.spinningspecies.com/davidia.html.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230046&req=5

Figure 3: Twelve representative images from the time-lapse videos (Video S1, S2), showing the development of a male FU, from flower (A–D) and stamen initiation (EF) to anther development (G–K). Due to meristem growth the magnification was changed between (A–F) (Video S1) and (G–L) (Video S2). The corresponding bar lines are indicated in (A,G). The white and black arrows mark two individual primordia at different developmental states. The outer primordia (black arrow) are clearly initiated later than the inner ones (white arrow). Videos available at www.spinningspecies.com/davidia.html.
Mentions: Since common binocular microscopes have angular optical paths that produce a shift of images in a stack, we used a monocular microscope (Leitz Wetzlar, Figure 2). Photographs were taken with a Canon Powershot G9 that was mounted on the eyepiece and triggered by a computer (Canon CameraWindow). For an automated stacking procedure a stepper motor was adapted to the focus wheel of the microscope and controlled by a four-channel motor driver circuit (L298N). For illumination a LED light was switched on during the image capture process. The whole stacking cycle was controlled by an open-source microcontroller (Arduino Leonardo) that was connected to the motor driver to change the focus. The camera was triggered by an implemented keyboard source code via a computer and the LED was controlled via a relay. Each cycle began with the activation of the LED and was followed by raising the microscope to the highest focus point. The next two steps that triggered the camera and moved the microscope to a lower focus position were repeated until the lowest focus point was reached (70 times). To finish the cycle the LED was turned off. For live imaging the cycle was triggered and repeated once per hour by a timer (remote control for cameras). Since the meristem grew, the focus range was checked twice a week and manually readjusted. After the meristem became too large the magnification was reduced from a 10-fold to a 4-fold objective (comp. Figures 3A–F and G–L). Accordingly, live observation is divided into two time lapse videos.

Bottom Line: The growing meristem was observed for 30 days, the longest live observation of a meristem achieved to date.D. involucrata exemplarily shows that live-ELM gives new insights into developmental processes of plants.In addition to morphogenetic questions such as the transition from vegetative to reproductive meristems or the absolute timing of ontogenetic processes, this method may also help to quantify cellular growth processes in the context of molecular physiology and developmental genetics studies.

View Article: PubMed Central - PubMed

Affiliation: Institut für Spezielle Botanik, Johannes Gutenberg-Universität Mainz, Germany.

ABSTRACT
Morphogenesis in plants is usually reconstructed by scanning electron microscopy and histology of meristematic structures. These techniques are destructive and require many samples to obtain a consecutive series of states. Unfortunately, using this methodology the absolute timing of growth and complete relative initiation of organs remain obscure. To overcome this limitation, an in vivo observational method based on Epi-Illumination Light Microscopy (ELM) was developed and tested with a male inflorescence meristem (floral unit) of the handkerchief tree Davidia involucrata Baill. (Nyssaceae). We asked whether the most basal flowers of this floral unit arise in a basipetal sequence or, alternatively, are delayed in their development. The growing meristem was observed for 30 days, the longest live observation of a meristem achieved to date. The sequence of primordium initiation indicates a later initiation of the most basal flowers and not earlier or simultaneously as SEM images could suggest. D. involucrata exemplarily shows that live-ELM gives new insights into developmental processes of plants. In addition to morphogenetic questions such as the transition from vegetative to reproductive meristems or the absolute timing of ontogenetic processes, this method may also help to quantify cellular growth processes in the context of molecular physiology and developmental genetics studies.

No MeSH data available.