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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we describe an improvement of the semi-in vitro (SIV) method for observing pollen tube guidance and reception in Arabidopsis thaliana, which increases the receptivity of ovules. The high-throughput SIV cum septum method may be coupled with gametophyte marker lines and genetically encoded biosensors to monitor the dynamic process of fertilization.

Abstract

In flowering plants, the growth and guidance of the pollen tube (male gametophyte) within the pistil and the reception of the pollen tube by the female gametophyte are essential for double fertilization and subsequent seed development. The interactions between male and female gametophytes during pollen tube reception culminate in pollen tube rupture and the release of two sperm cells to effect double fertilization. As pollen tube growth and double fertilization are deeply hidden within the tissues of the flower, this process is difficult to observe in vivo.

A semi-in vitro (SIV) method for the live-cell imaging of fertilization in the model plant Arabidopsis thaliana has been developed and implemented in several investigations. These studies have helped to elucidate the fundamental features of how the fertilization process occurs in flowering plants and which cellular and molecular changes occur during the interaction of the male and female gametophytes. However, because these live cell imaging experiments involve the excision of individual ovules, they are limited to a low number of observations per imaging session, making this approach tedious and very time-consuming. Among other technical difficulties, a failure of the pollen tubes to fertilize the ovules in vitro is often reported, which severely confounds such analyses.

Here, a detailed video protocol for the imaging of pollen tube reception and fertilization in an automated and high-throughput manner is provided, allowing for up to 40 observations of pollen tube reception and rupture per imaging session. Coupled with the use of genetically encoded biosensors and marker lines, this method enables the generation of large sample sizes with a reduced time investment. Nuances and critical points of the technique, including flower staging, dissection, medium preparation, and imaging, are clearly detailed in video format to facilitate future research on the dynamics of pollen tube guidance, reception, and double fertilization.

Introduction

The generation of genetically unique offspring in sexually reproducing organisms is dependent on the successful fusion of male and female gametes. In flowering plants, the interaction of two male gametes (sperm cells) with two female gametes (egg cell and central cell) during double fertilization depends on sperm release from the pollen tube (the male gametophyte). This process, called pollen tube reception, is largely controlled by the synergid cells that reside within the embryo sac (the female gametophyte)1,2. As pollen tube reception takes place deep inside the flower, a method allowing for live-cell imagi....

Protocol

NOTE: See the Table of Materials for a list of the materials and equipment used in this protocol.

1. Considerations for designing the imaging experiment

  1. Predetermine the imaging duration and sampling frequency required to capture the desired phenomenon to be observed. For example, following Nyquist's sampling theorem, the sampling frequency must be greater than twice the highest frequency of the input sample. Therefore, to accurately capture sy.......

Representative Results

To assess the timing of nuclear degeneration in the receptive synergid with respect to pollen tube rupture in Arabidopsis, as well as to observe whether the left or right synergid is predestined to become the receptive synergid, the SIV cum septum method described here was employed using a female gametophyte nuclear marker stacked with a synergid cytosolic marker (pFG:roGFP2-ORP1-NLS, pMYB98:roGFP2-ORP1) as the septum donor and a male gametophyte marker (pLAT52:R-GECO) as the poll.......

Discussion

This manuscript introduces an efficient protocol for the imaging of pollen tube reception and double fertilization in Arabidopsis. The improved method, SIV cum septum, greatly increases the percentage and total number of successful pollen tube reception events that are observable per imaging session. The representative results shown here demonstrate an imaging session with 41 successful pollen tube reception events and 10 ovules showing reception defects (~80% efficiency). This is over double the n.......

Acknowledgements

We thank Sara Simonini and Stefano Bencivenga for donating the pFG:roGFP2-ORP1-NLS construct and Christof Eichenberger, Johann Almendinger, Vincent Sutter, and Celia Baroux for their advice on microscopy. We kindly acknowledge advice from Ravi Palanivelu, Philipp Denninger, Sharon Kessler, Mark Johnson, Tomokazu Kawashima, and everyone else at the International Conference on Sexual Plant Reproduction 2022 who showed interest in a protocol on SIV cum septum. This work was supported by the University of Zurich and grants from the Swiss National Science Foundation to U.G.

....

Materials

NameCompanyCatalog NumberComments
1 mm glass slide Epredia 16211551
35 mm glass bottom dish (14 mm well)Mattek P35G-1.5-14-C
Calcium Chloride Roth CN93.1
Columbia (Col-0) Nottingham Arabidopsis Stock Centre (NASC)stigma donor
Dissecting Scope Olympus SZX2-ILLT
Insulin needle (0.3 G)BD304000
Landsberg erecta (Ler-0) Nottingham Arabidopsis Stock Centre (NASC)septum donor
Magnesium SulfateMerck5886
Potassium ChlorideRoth6781.1
Razor bladeBeldura7026797
Scotch double sided tape Scotch768720Less thick and good for stigma dissection
Sodium Chloride Roth3957.1
SucroseITW reagentsA2211,1000
Tesa double sided tape Tesa05681-00018Very sticky and good for septum dissection
Ultra low gelling temperature agaroseFMC SeaPrep50302

References

  1. Huck, N., Moore, J. M., Federer, M., Grossniklaus, U. The Arabidopsis mutant feronia disrupts the female gametophytic control of pollen tube reception. Development. 130 (10), 2149-2159 (2003).
  2. Rotman, N., et al.

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