Method for determination of pollen viability and/or maturation grade of a pollen population
Inventors
Heidmann, Iris • Di Berardino, Marco • Kok, Kim • Schade-Kampmann, Grit
Assignees
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Publication Number
US-9797855-B2
Publication Date
2017-10-24
Expiration Date
Abstract
A method for the determination of pollen viability and/or maturation grade of a pollen population, comprising mechanically removing pollen grains from flowers; re-suspending pollen grains in an electrically conductive buffer for keeping the plant cells to remain viable; passing the pollen suspension through an appropriate filter with a pore size suitable for a microfluidic device being adapted to perform impedance flow cytometry (IFC); An advantage of the present invention is that a plant cell does not have to be stained for viability analysis, the method is non-invasive and a high number of cells can be analyzed in a short time frame in real-time and on-site. It is possible to follow developmental processes of plant cells, and that it is applicable to all plant species. In particular, the method allows a standardized measurement independent of the location and all over the world.
Core Innovation
The invention provides a label-free, non-invasive on-site microfluidic impedance flow cytometry method for determining at least one of pollen viability and maturation grade of a pollen population. Immature or mature pollen grains are mechanically removed from flowers and re-suspended in an electrically conductive, specific and well-defined buffer for keeping the pollen grains to remain viable.
A cell suspension with the pollen grains is passed through a sieve with an appropriate mesh size suitable for a microfluidic device adapted to perform impedance flow cytometry by providing an alternate current field to form a filtered suspension. The filtered suspension is passed through the microfluidic device calibrated for a respective pollen type, and an impedance of the pollen grains is measured at predetermined frequencies.
Measured impedance values of each pollen grain are stored with an amplitude value and its correlated phase angle as well as the predetermined frequencies. The pollen grains are discriminated according to the amplitude or phase values by manually or automatically setting an appropriate gate to determine a viable to non-viable ratio or maturation grade, and the stored impedance values can be arranged in an x-y matrix to identify viability and determine a gate between data groups having a lower and a higher phase angle.
The method further supports identifying maturation grade by determining at least one of the amplitude values and the angle values as a function of the measurement frequencies. The amplitude values and the angle values are compared with values of a database comprising data of respective pollen, and pollen grains are identified as belonging to a specific developmental stage, with the result displayed for the analysed population.
Claims Coverage
The partial content includes two independent claims. Across these claims, the inventive features focus on microfluidic IFC measurement of individual pollen grains, storage of amplitude/phase-angle impedance data at predetermined frequencies, and classification using gating and database comparison to derive a viable-to-non-viable ratio and maturation/developmental-stage assignments.
Microfluidic IFC pollen suspension preparation and filtration
Mechanically removing immature or mature pollen grains from flowers; re-suspending the pollen grains in an electrically conductive, specific and well-defined buffer for keeping the pollen grains to remain viable; passing a cell suspension with the pollen grains through a sieve with an appropriate mesh size suitable for a microfluidic device adapted to perform impedance flow cytometry by providing an alternate current field to form a filtered suspension.
Calibrated microfluidic impedance measurement at predetermined frequencies
Passing the filtered suspension through the microfluidic device calibrated for a respective pollen type and measuring an impedance of the pollen grains at predetermined frequencies.
Per-grain impedance storage with amplitude and correlated phase angle
Storing measured impedance values of each pollen grain with an amplitude value and its correlated phase angle as well as the predetermined frequencies; counting the pollen grains in the suspension.
Amplitude/phase gating to yield viable-to-non-viable ratio or maturation grade
Discriminating the pollen grains according to the amplitude or phase values by manually or automatically setting an appropriate gate and determining a viable to non-viable ratio or maturation grade of the analysed pollen population.
x-y-matrix arrangement with phase-angle gate and database ratio comparison
Identifying whether the pollen grains are viable or non-viable by arranging the stored impedance values with the amplitude value and the phase angle value in an x-y-matrix; determining a gate between data groups having a lower and a higher phase angle; counting the pollen grain impedance values of different data groups; calculating a ratio between the amount of pollen having an impedance value with a phase angle below the gate and the amount of pollen having an impedance value with a phase angle above the gate; comparing the ratio with a respective ratio of a database comprising data of respective pollen for determining whether the viability of the pollen is sufficient for a given certain purpose; displaying the result for that analysed population.
Frequency-dependent amplitude/angle comparison for maturation grade and developmental stage
Identifying the maturation grade by determining at least one of the amplitude values and the angle values as a function of the measurement frequencies; comparing the amplitude values and the angle values with the respective values of a database comprising data of respective pollen; identifying the plant cells as belonging to a specific developmental stage; and displaying the result for that analysed population.
Across the independent claims, the core claim coverage centers on microfluidic IFC of individual pollen grains with impedance measured at predetermined frequencies, stored per grain as amplitude and correlated phase angle, and classified using an appropriate gate and database comparisons to provide viability, including a viable-to-non-viable ratio, and maturation grade or developmental-stage identification.
Stated Advantages
Staining-free pollen viability and/or maturation grade determination.
Real-time high-throughput analysis.
Standardized and location-independent measurement.
Label-free, non-invasive, on-site, non-destructive analysis.
Documented Applications
Male fertility/sterility selection.
Seed production control.
Detection of stress, pesticide, and insect effects.
Germplasm preservation/pollen gene bank.
Contamination detection, including bacterial, fungal, or other contaminations in seed production/plant cell production processes.
Examples of correlations to seed setting and embryo yield (tomato).
Assessing viability changes across plant age and CMS lines (Raphanus).
Differentiation of pollen developmental stages (Brassica).
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