Abstract
The development of sperm cells (SCs) from microspores involves a set of finely regulated molecular and cellular events and the coordination of these events. The mechanisms underlying these events and their interconnections remain a major challenge. Systems analysis of genome-wide molecular networks and functional modules with high-throughput “omics” approaches is crucial for understanding the mechanisms; however, this study is hindered because of the difficulty in isolating a large amount of cells of different types, especially generative cells (GCs), from the pollen. Here, we optimized the conditions of tomato pollen germination and pollen tube growth to allow for long-term growth of pollen tubes in vitro with SCs generated in the tube. Using this culture system, we developed methods for isolating GCs, SCs and vegetative cell nuclei (VN) from just-germinated tomato pollen grains and growing pollen tubes and their purification by Percoll density gradient centrifugation. The purity and viability of isolated GCs and SCs were confirmed by microscopy examination and fluorescein diacetate staining, respectively, and the integrity of VN was confirmed by propidium iodide staining. We could obtain about 1.5 million GCs and 2.0 million SCs each from 180 mg initiated pollen grains, and 10 million VN from 270 mg initiated pollen grains germinated in vitro in each experiment. These methods provide the necessary preconditions for systematic biology studies of SC development and differentiation in higher plants.
Highlights
During the development of sperm cells (SCs, male gamete) from microspores in higher plants, the microspore generated from diploid microsporocytes via meiosis first undergoes asymmetric mitosis to produce a larger vegetative cell (VC) and a smaller generative cell (GC) embedded in the VC
We developed efficient protocols to isolate a large amount of GCs, SCs, and vegetative cell nuclei (VN) at high purity to satisfy the demands of “omics” study
Dynamics of GCs and SCs During Culture In Vitro Our experiments showed that low-temperature (−20◦C) stored tomato pollen grains without prehydration germinated in vitro at low germination rate (Supplementary Table S1)
Summary
During the development of sperm cells (SCs, male gamete) from microspores in higher plants, the microspore generated from diploid microsporocytes via meiosis first undergoes asymmetric mitosis to produce a larger vegetative cell (VC) and a smaller generative cell (GC) embedded in the VC. Depending on the plant species, GC mitosis occurs before anthesis or in growing pollen tubes; released mature pollen at anthesis is tricellular in some species such as Oryza sativa, Zea mays, and Arabidopsis thaliana (Berger and Twell, 2011) or bicellular in other species such as Lilium brownii and Solanum lycopersicum. Systematic “omics” studies of the development process are essential for understanding the mechanisms
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