A plant breeder must impose selection based on a reliable evaluation of hundreds and then dozens of different genotypes to identify breeding lines that are an improvement over what a farmer currently grows.  Thus, HTP must go through rigorous testing if it will replace or add to the breeder’s current evaluation plan. One part of this research process is called ground-truthing. Ground-truthing is exactly what it sounds like: the data collected manually at the field location is assumed to be a true measurement of the plant’s phenotype. The data collected by the sensors on the UAV can be compared with the data collected by the normal field methods. Ground-truthing then is the process of determining if a particular, repeatable method of analyzing the sensor data ‘lines up’ with the differences measured with hands on methods. Ground-truthing is how breeders know UAVs are accurate, because this data collection method is used as the standard to which data collected from high throughput phenotyping technologies is compared using statistical analysis. Collecting ground-truth data can be time consuming and physically demanding. Fortunately, as advances are made in high throughput phenotyping technologies, we can phase out the need for ground-truthing. 

In current programs, ground-truthing is used to measure traits which plant breeders categorize into one of two groups: basic traits and leaf-level traits. Basic traits are those we can see with our eyes which allow us to perform a visual or manual measurement to collect data. Examples of basic traits include flowering time, emergence, and plant weights.

Figure 13. Height measure in a plot. This is a single measurement. Several more will need to be completed to ground-truth or to calculate average plant height for cultivar selection.

Captured by C. Mick, 2022.

One basic trait of interest to breeders is height. Breeders are interested in plant height because it is correlated with plant health. Plant height is also being used to predict final yields of both yield and biomass. Plant height is one of the simplest traits to gather ground truth data on because of how it is collected. To gather height data, researchers will go out into the field with a meter stick and clipboard. In the case of height, researchers had to measure the height enough times in enough environments to get an average (Fig. 13). They had to physically walk out into a field multiple times in the summer to get an average plant height. Based on these measurements they then selected the best breeding lines to move forward in the breeding process. Repeat this process over the course of 5-10 years and you have a cultivar.

Leaf level traits require more effort to score because these traits are not reliably detectable to the human eye. Leaf level traits such as chlorophyll concentration and light reflectance can be measured with hand-held sensors. Breeders are interested in chlorophyll concentration and leaf-level light reflectance because they are strong indicators of plant health. The leaf area index (LAI) can also be measured by sensors. LAI is the ratio of leaf area per unit of ground. Healthy plants can grow at different rates so using leaf level measurements to quantify the amount of vegetation in a specific area of the plot is an important trait.

Measuring leaf area index can be done either directly or indirectly. Direct measurements are generally more accurate, but it comes at the price of being highly destructive. One way direct measurements are done is by harvesting all the leaves in a defined area and measuring the area of each leaf to determine the total. This is classified as destructive sampling because the plants are removed from the plot to collect the data. Another method is doing litterfall collection. This is done by collecting fallen leaves in traps and calculating leaf area index by multiplying dry mass by the specific leaf area. This process is done over a period of time to calculate leaf area index. There are two major downfalls to the second method. The first is that it is labor intensive. The litter fall traps must be frequently emptied to avoid decomposition and biomass loss. The second is that the specific leaf area used in calculations is only an estimate, so there may be some inaccuracy.

Indirect measurements have a slight edge over direct measurement methods because they are nondestructive and not as time-consuming. Indirect measurements are possible because recent research has developed ways to correlate these with direct measurements. For biomass specifically, research has shown that leaf area index can be a good indicator of biomass. The bonus: it is completely nondestructive. To measure leaf area index, we use plant canopy analyzers called ceptometers (Fig. 14).  A ceptometer measures photosynthetically active radiation (PAR). To measure leaf area index, a thin, long sensor called a probe is placed underneath the plant canopy to measure the amount of PAR that travels through the canopy. Simultaneously an external sensor is held above the plant canopy to measure the amount of PAR coming in (Fig. 15). Both measurements are used to calculate the amount of PAR that is blocked by the canopy which correlates to the amount of leaf cover and thus leaf area index.

Figure 14. A ceptometer. The main parts of the sensor include the probe, display, keypad, and external sensor. Samples are collected by placing the probe under the canopy and the external sensor above the canopy. Data points are recorded and annotated using the keypad and seen in the display.

Captured by C. Mick, 2022.

Figure 15. Using a ceptometer in the field. (Left) This is a two-person job where one person holds the external sensor above the canopy and the other person holds the probe under the canopy. (Right) The entire length of the probe takes light readings to measure PAR.

Captured by L. Wang, 2022.