Abstract
Key messageAn easy and highly reproducible nondestructive method named the Leaf Collar Method is described to identify and characterize the different stages of pollen development in maize.In plants, many cellular events such as meiosis, asymmetric cell division, cell cycle regulation, cell fate determination, nucleus movement, vacuole formation, chromatin condensation and epigenetic modifications take place during pollen development. In maize, pollen development occurs in tassels that are confined within the internal stalk of the plant. Hence, identification of the different pollen developmental stages as a tool to investigate above biological processes is impossible without dissecting the entire plant. Therefore, an efficient and reproducible method is necessary to isolate homogeneous cell populations at individual stages throughout pollen development without destroying the plant. Here, we describe a method to identify the various stages of pollen development in maize. Using the Leaf Collar Method in the maize inbreed line B73, we have determined the duration of each stage from pollen mother cells before meiosis to mature tricellular pollen. Anther and tassel size as well as percentage of pollen stages were correlated with vegetative stages, which are easily recognized. The identification of stage-specific genes indicates the reproducibility of the method. In summary, we present an easy and highly reproducible nondestructive method to identify and characterize the different stages of pollen development in maize. This method now opens the way for many subsequent physiological, morphological and molecular analyses to study, for instance, transcriptomics, metabolomics, DNA methylation and chromatin patterns during normal and stressful conditions throughout pollen development in one of the economically most important grass species.
Highlights
The dissection of male gametophyte development provides a unique tool to study many fundamental questions in plant cell biology and reproduction
The events that culminate in the formation and release of mature pollen grains from anthers initiate with the formation of diploid pollen mother cells followed by meiosis generating four microspores, which further develop into pollen grains harboring one and two haploid sperm, respectively, enclosed by the vegetative tube cell (Figure S1)
Variation of plant height and vegetative (V) stage transitions of maize plants staged by the Leaf Collar Method
Summary
The dissection of male gametophyte (pollen) development provides a unique tool to study many fundamental questions in plant cell biology and reproduction. Microspores as one stage during pollen development represent the major source to generate di-haploid plants and are key to modern agriculture. Pollen development in anthers involves intricate and tightly controlled sets of structural and molecular changes and is characterized by a relatively synchronized gene expression pattern Previous reports have shown a correlation between anther length and pollen development (Chang and Neuffer 1989), but plants had to be dissected to isolate anthers. Implementing an effective method of correlating vegetative plant development with pollen stages is fundamental for an accurate selection of material for gene expression analyses, comparative genomics and physiology, as well as investigations of pollen development under stress
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