**4. High-throughput plant phenotyping platforms and chlorophyll fluorescence techniques**

The advancement of plant phenotyping is a key factor for the success of modern plant breeding and basic plant research. Since the recognition of the phenotyping bottleneck to plant breeding [12], a global effort to provide HTPP platforms was set on. Currently, most platforms are userbuilt, but some commercial platforms are already available, as is the case of the German platform from the manufacturer LemnaTec and the Czech platform from the manufacturer Photon System Instruments (PSI). The Canadian-based firm Qubit Systems offers a modified version of the PSI platform. To provide high-throughput phenotyping capabilities to plant breeders, numerous user-built phenotyping facilities are organized in networks, the most prominent being the European Plant Phenotyping Network, which offers access to 23 different plant phenotyping facilities to the user community [116]. Some countries organized national networks, as is the case of the German Plant Phenotyping Network [117] and the UK Plant Phenomics Network [118]. The Jülich Plant Phenotyping Centre [119] is a leading EPPN light-adapted samples), which measures the effective quantum yield of PS II [96], PAM fluorometry allows the construction of rapid light curves (RLCs) relating the rate of photo‐ synthetic electron transport and incident photon irradiance [97, 98]. PAM was successfully applied to a wide range of plants, such as the olive tree, rosemary, and lavender [99]; *Paspalum dilatatum* [100], *Phillyrea angustifolia* [101], and other Mediterranean shrubs [102]; the tropical grass *Setaria sphacelata* [84] and several C4 turfgrasses [103]; maize [36, 104]; and *Arabidopsis*

The development of Chl fluorescence imaging systems by numerous research groups [106– 108] together with the emergence of commercially available models by PSI (Brno, Czech Republic), Walz Systems (Effeltrich, Germany), and Technologica Ltd. (Colchester, UK) has greatly increased the versatility of Chl fluorometry techniques (reviewed in [109]). Systems that image at the microscopic level allow to measure PS II photochemical efficiencies from chloroplasts within intact leaves and from individual cells within mixed populations [107, 110]. On the other hand, lower resolution imaging systems allow the mapping of fluorescence parameters over large areas, making it a unique technique to study the spatial heterogeneity of the photosynthetic activity across an autotrophic surface [111–113]. Conventional and imaging techniques use different technologies, namely in the detection processes of the fluorescence signal: a photodiode or phototube in conventional PAM fluorometry and a charge-coupled device (CCD) camera in imaging-PAM fluorometry. Consequently, caution is needed when comparing results from conventional and imaging fluorescence techniques [114]. Nevertheless, imaging-PAM fluorometry has proven to be a powerful technique and new technological developments, as the use of semi-automated systems equipped with fluores‐ cence cameras continuously assessing the photochemical activity of leaves [115] are in course.

**4. High-throughput plant phenotyping platforms and chlorophyll**

The advancement of plant phenotyping is a key factor for the success of modern plant breeding and basic plant research. Since the recognition of the phenotyping bottleneck to plant breeding [12], a global effort to provide HTPP platforms was set on. Currently, most platforms are userbuilt, but some commercial platforms are already available, as is the case of the German platform from the manufacturer LemnaTec and the Czech platform from the manufacturer Photon System Instruments (PSI). The Canadian-based firm Qubit Systems offers a modified version of the PSI platform. To provide high-throughput phenotyping capabilities to plant breeders, numerous user-built phenotyping facilities are organized in networks, the most prominent being the European Plant Phenotyping Network, which offers access to 23 different plant phenotyping facilities to the user community [116]. Some countries organized national networks, as is the case of the German Plant Phenotyping Network [117] and the UK Plant Phenomics Network [118]. The Jülich Plant Phenotyping Centre [119] is a leading EPPN

*thaliana* [105], among others.

10 Applied Photosynthesis - New Progress

**fluorescence techniques**

*3.2.3. Imaging pulse amplitude modulated fluorescence*

member. Jülich's platform makes use of LIFT to perform middle-range remote sensing of crops [119]. The commercial phenotyping platforms do not use LIF technologies. A simplified JIP test (restricted to the calculation of *F*v/*F*m) is used in the commercial platform from LemnaTech; a LemnaTech module with this feature is incorporated in the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) HTPP Platform [120]. PlantScreen, the commercial platform from PSI, uses conventional PAM fluorometry to perform quenching analysis. This platform has been used to phenotype cold-tolerant pea (*Pisum sativum*) plants [121]. Kjaer and Ottosen [122] used six independent PAM fluorometers in a HTPP experiment to assess daily growth of field-grown *Brassica napus*. In this case, however, the PAM system was exclusively used to show that the near infra-red laser beam of a 3D laser scanning, used for phenotyping, had not a deleterious effect over the photosynthetic metabolism of the plants. Bellasio and coworkers [123] have used an imaging-PAM system based on a FluorCam camera (PSI) inserted in a userdeveloped setting to phenotype common bean (*Phaseolus vulgaris*). The French platforms based at Montpellier [124] use an imaging-PAM system based on Walz's devices. An important advantage of Chl fluorescence imaging is that it can be used to screen a large number of small plants simultaneously [125]. A recent advance introduced by Serôdio and coworkers [126] allows the rapid generation of light curves from non-sequential, temporally independent fluorescence measurements. This technique has the potential to bring the valuable information provided by fluorescence RLCs into the realm of HTPP. David Kramer's group, at the Michigan State University, is currently developing a multi-instrument platform entitled Dynamic Environmental Photosynthetic Imaging (DEPI) [127], with the aim of reproducing in phyto‐ trons the dynamics of field conditions, while continuously recording multiple parameters related with them photosynthetic performance.



Note: JPPC: Jülich Plant Phenotyping Centre [119]; IPK: Leibniz Institute of Plant Genetics and Crop Plant Research [120]; M3P: INRA—Montpellier Plant Phenotyping Platforms [124].

**Table 1.** Applications of chlorophyll a fluorescence techniques in high-throughput plant phenotyping.
