Label-free optical interferometric microscopy to characterize morphodynamics in living plants

During the last century, fluorescence microscopy has played a pivotal role in a range of scientific discoveries. The success of fluorescence microscopy has prevailed despite several shortcomings like measurement time, photobleaching, temporal resolution, and specific sample preparation. To bypass these obstacles, label-free interferometric methods have been developed. Interferometry exploits the full wavefront information of laser light after interaction with biological material to yield interference patterns that contain information about structure and activity. Here, we review recent studies in interferometric imaging of plant cells and tissues, using techniques such as biospeckle imaging, optical coherence tomography, and digital holography. These methods enable quantification of cell morphology and dynamic intracellular measurements over extended periods of time. Recent investigations have showcased the potential of interferometric techniques for precise identification of seed viability and germination, plant diseases, plant growth and cell texture, intracellular activity and cytoplasmic transport. We envision that further developments of these label-free approaches, will allow for high-resolution, dynamic imaging of plants and their organelles, ranging in scales from sub-cellular to tissue and from milliseconds to hours

Sampling rate in the dynamic speckle analysis

Dynamic laser speckle and its biological version (biospeckle laser) have been used in many areas of knowledge. Its noninvasive approach allows the application in advantage regarding those that need contact or damage the analyzed sample. However, one needs the sharp adjust of the image acquiring and processing. In this article, we show how the variation of sampling rate in a dynamic speckle analysis affects the value of dynamic speckle indexes concerning the absolute value of the differences index, the temporal speckle standard deviation index, and the temporal speckle mean index. We show that the dynamic speckle index value changes its maximum excursion with the variation of sampling rate, affected directly by the camera's time integration (time of exposure). We highlight the importance of knowing the frequency band of the analyzed phenomenon and its signal to choose the appropriate sampling rate, with the recommendation of using the lowest sampling rate possible¿without compromise the speckle grains¿to obtain an acceptable maximum excursion and an illumination level with a good signal-noise ratio. The results will help those who work with the phenomenon/technique to enhance their analysis tailoring the set up and yielding reliable results, since the optical method demands a rigorous bias of the image acquiring and processing

A study of the physiology and genetics of rapid rooting traits in lettuce (Lactuca sativa)

Lettuce is usually germinated and grown for a short period in nurseries before planting (transplanted) in the field in the UK and Europe.Plants that are transplanted are more uniform in maturity due to more uniform germination, avoid early environmental stresses and usually mature earlier than direct sown crops. Lettuce transplants need to establish quickly in the field to optimise growth and uniformity and can be exposed to stresses that include mild initial drought,variable soil and environmental conditions and root pruning. These stresses are only likely to be exacerbated with increasing pressures on growers to reduce inputs. Identification of phenotypic and genotypic variation for a “rapid rooting”trait in the lettuce gene pool therefore may enable faster establishment and has the potential to improve the performance of lettuce transplants if integrated into a marker assisted breeding programme for lettuce varieties.The following work optimised a 2D high-throughput assay to screen 14-dayold seedlings of a lettuce diversity fixed foundation set (DFFS)and identified phenotypic variation for key rapid rooting traits that could prove important for future breeding programmes. Phenotypic variation was also observedwithin the DFFS fordeeper rooting potential of lateral rootsandfor root hair traits.The 2D assay was also utilised to identify 16 significant quantitative trait loci (QTL) associated withthe rapid rooting phenotype, of which six were associated with increased primary root growth, three with increased lateral root growth, two were associated with lateral root length density, three with lateral root number density and two with mean lateral root length. Atargeted transcriptomic analysis utilising extreme lines identifiednine candidate genes located under five of the reported QTL for the “rapid rooting” phenotype. The genes coded for proteins involved in various pathways involved with root growth including cell proliferation, cell expansion, cell wall synthesis and ABA synthesis. Thesegenes mayoffer a promising approach for the improvement of lettuce establishment in a commercial breeding programme.The extreme lines were then analysed in a 3D transplant sand assay to assess the effect of altering the root:shoot ratio through controlled root pruninghad on the rapid rooting phenotype and identified that although some ofthelines behaved differently some of the lines maintain a rapid rooting phenotype at transplant maturity and recovered a larger proportion of the root:shootratio compared to the slower rooting lines and commercial controls