AstroPlant Growth Protocol
The scientific purpose of the AstroPlant experiments is to provide data on plant growth and development in various environmental conditions. This data is used to develop and calibrate a general plant growth model to help inform farming decisions in space and on earth. Only if the plants of the same species in all AstroPlant kits are grown the same, will we be able to compare them and the influence of their environments.
As species often react differently to the environment, the model will contain species-specific variables. This makes it necessary to conduct experiments with a lot of species in a myriad of varying conditions. Some of the most important agricultural crops are also the focus of ESA’s MELiSSA program, which aims to develop a Closed Ecological Life Support System (CELSS) incorporating higher plants. Therefore, wheat (Triticum aestivum L.) and soybean (Glycine max L.) have been selected as crops of interest for AstroPlant experiments.
In order to allow for shorter duration experiments while also providing data on a different type of crop, basil (Ocimum basilicum L.) has also been selected. In addition, basil experiments allow for more rapid validation of the protocol and kit design. This is because basil is a crop that can grow rapidly under the right circumstances, but will wilt (i.e. show hanging and dried up leaves) quickly if the there is something wrong.
If you choose to conduct an experiment with settings different from those of the protocol, you need to indicate this in detail when reporting the manual measurements. This is critical for the right interpretation of your results!
When placing the AstroPlant kit, it is important to:
- Place it indoors so the temperature at night does not (regularly) fall below 10 °C;
- Place it on a level and elevated surface;
- Place it in a room without irregular temperature changes.
- Not place it where there is very little or a lot of air movement;
- Not place it in very dry (RH <40%) or very humid conditions (RH >90%);
- Not place it directly in sunlight.
- Consider the noise produced by the pump
- Place it in a room with wi-fi connection
A Dutch bucket hydroponic system, "WaterFarm" from General Hydroponics Europe (GHE), has been selected to supply the roots with water and nutrients. Clay pebbles known as "Lightweight Expanded Clay Aggregate" (LECA) will be used as the substrate (fig.1). Every time before starting a new experiment, the LECA needs to be rinsed several times to ensure that no nutrients remain from the previous experiment.
- 7 Liters of LECA clay small pebbles is enough to fill the root compartment
- 18 Liters of nutrient solution (diluted) per experiment is needed
See 8-A for more information on acquiring the right nutrient solution and substrate.
Figure 1. LECA (lightweight expanded clay aggregate).
- 1.
- 2.Rinse the clay pebbles with regular tap water until they are free of dust (at least 3 times).
- 3.Fill the root compartment with the clay pebbles.
- 4.Fill the nutrient solution reservoir with 6 litres of 7-4-10 NPK fertiliser solution.
- 5.Place the hydroponic system inside the AstroPlant kit.
- 6.Replace the nutrient solution in the kit every two weeks.
Only mix the amount of nutrient solution you need! If the solution stays on the shelf for four days, some nutrients can already precipitate.
Basil grows well with a 16 hour photoperiod (i.e. 16 hours of light, 8 hours of darkness every day). Therefore, the ON time for all LED lights needs to be set to 06:00 – 22:00 (6 am – 10 pm). The intensity of the LED lights need to be set on: 10% for red, 4% for blue and 2,5% for far red. You can check this in the configuration menu.
Seeds provided by AstroPlant are supplied by a horticultural company for use in the agribusiness. They will have a very high germination rate (80% - 99%, depending on the species). Therefore, it is not be necessary to select the ‘better’ seeds beforehand, and every planted seed can be expected to grow normally.
If the seeds you plan to use are not supplied by a horticultural company for use in agribusiness, it can help your experiment if you make a selection of the ‘better’ seeds before planting. Seed characteristics differ a lot between species, but there are some common traits that can be checked:
- Seeds that differ from the morphology of the majority of the seeds in the package, are usually less viable.
- Larger seeds are usually stronger and have higher germination rates.
- Seeds need to have an intact seed coat in order to allow for proper imbibition and defence against pathogens.
If you are using a self-selected species or cultivar, check to see if the species you wish to grow requires a period of vernalisation (cold storage). This is commonly noted on industry supplied packages, as it has a large impact on the germination rate and germination time.
Before starting the experiment, it is crucial to sterilise the seeds. Especially seeds from non-industry sources often have spores of pathogens on them, that will contaminate the experiment right from the start.
- 1.Immerse the seeds in 70% ethanol for 2 minutes.
- 2.Prepare a 20% commercial bleach solution with a drop of common detergent.
- 3.Place the seeds in 10 ml of this solution for 15 minutes, swirling gently from time to time (or use a magnetic stirring rod in the beaker).
- 4.Rinse the seeds four times with distilled and sterilised water.
Due to the rough matrix of the clay pebbles, the basil seeds need to be germinated outside of the kit. It is best to prepare 2 seedlings per kit if the seeds were sent from AstroPlant (for those have 85% germination rate). If you have acquired your own basil seeds, it is best to prepare 5 seedlings per kit, as they will have a lower germination rate. This way, you will certainly have a viable seedling to start the experiment with. Place the seeds with wet filter paper in a petri dish or small tube. After 8-12 days in the dark, they will have germinated.
Make sure the seeds stay sufficiently wet throughout those 8-12 days.
Check daily to see if the seeds have germinated and both the radicle (first part of the root) and the hypocotyl (first part of the stem) are visible. If this is the case, you can begin planting.
- 1.Take the root compartment out of the AstroPlant kit to get better access.
- 2.Select the best seedling and plant only this one. Seedlings with unusually long stems and bleak leaves, and those with brownish or curled-up roots should not be used.
- 3.Take a few clay pebbles out of the root compartment and place the seedling inside, 1-2 cm from the irrigation ring. Carefully use a pincette for the placement, and only touch the stem of the seedling.It is not crucial that the radicle and hypocotyl are precisely in the right direction; plants are very adaptable and will reorient themselves during growth. What is important, is that the seed remains near the surface and in the light.
- 4.Cover up the roots with the clay pebbles taken out before.
- 5.Place the root compartment back in the kit.
If you germinated seeds fall through the pebbles or fail to grow properly, you can instead grow your seedling on the filter paper until the first two true leaves are 0,5 cm or longer (see 4B; figure 5). After this, the plant should be sufficiently sturdy to find footing among the clay pebbles inside the AstroPlant kit. Because the roots will be larger, you need to handle the seedlings with great care!
If you keep growing the seedlings on the filter paper longer than just after emergence of the radicle and hypocotyl, do not forget to mention this in your manual measurements file (with the amount of days).
Basil is a bushy herb with fast growth. Because of this, and the cut-and-come-again harvesting common for basil, there is no developmental scale available. There are usually only three growth stages indicated; pre-flowering, full flowering and post-flowering. As basil is not grown for the harvest of its seeds or fruits, it is more useful to indicate basil growth in leaf area and fresh mass. Harvesting is usually possible in cycles from 40 days after seeding.
Under conditions better than open field, germination is expected between 8 and 12 days after sowing.[1] The results for basil growth shown below in table 1 and figure 2 were obtained under white LED light, but in soil rather than a hydroponic system. [2]
Table 1. Basil growth in soil under white LEDs. While not grown in a hydroponic system, this is a rough schedule of the standard growth of basil. From: Growth Rate of Sweet Basil and Lemon Balm Plants Grown Under Fluorescent Lamps and LED Modules (Frąszczak et al., 2014).
Figure 2. Typical greenhouse growth of basil. Representative pictures from 2 to 6 weeks of soil grown plants in the greenhouse during the fall. From: Light Quality Dependent Changes in Morphology, Antioxidant Capacity, and Volatile Production in Sweet Basil (Ocimum basilicum) (Carvalho et al., 2016).
Usually basil is harvested in cycles, with only some of the leaves being taken each time. This allows the same plant to grow new leaves again, which is more productive than harvesting the entire plant only once. However, the AstroPlant experiments are focused on plant growth, not optimal production strategy. To get the best information on how basil plants grow in an environment, they need to grow for six weeks after germination. Only if the basil plants in all AstroPlant kits are grown the same, will we be able to compare them and the influence of their environments.
When growing for six weeks in good conditions, your basil plant will start to flower (see figure 3). Because basil is grown for its leaves, the flowers need to be removed. This process is called ‘pruning’, and prevents the plant from directing resources to flower and seed development. Instead, after pruning, the plant will use those resources to develop more and larger leaves.
Figure 3. Two white flowers of basil. The flowers of basil remain quite small and reduce the growth of the leaves.
When performing the regular manual measurements, check to see if there are small white flowers starting to grow at the top of your basil plant. If you spot them, look from the top down along the main stem for the node with (small) leaves closest to the flower. A node is the point where a leaf or branch is attached to the main stem. Then, use scissors to cut the main stem 0,5 cm above this node (see figure 4).
Figure 4. Where to prune basil. At the top of the image, the green sepals of the flowers are visible. The first node below the flowers is indicated, 0,5 above which the stem should be cut. From: https://plantinstructions.com/herbs/how-to-prune-basil/
The goal of the AstroPlant experiments is to obtain an accurate and comprehensive dataset of how plants grow under different environmental conditions. Two types of measurements make up this dataset: automatic measurements and manual measurements. The automatic measurements are made by the various sensors in the AstroPlant kit and can be seen on the dashboard. While the data transfer is under development, we will ask you to send us your dashboard link so we can download the automatic measurement data you have collected and stored.
Manual measurements need to be conducted regularly during growth, and after harvest. Both are described in more detail in the next section. Currently, the AstroPlant app used for logging the manual measurements is still in development. Please use the ‘manual measurement’ excel files supplied with the kit to document your measurements.
Table 2. Automatic measurements of the plant.
Table 3. Automatic measurements of the environment.
* One hour after being switched on, or changed.
Table 4. Regular manual measurements.
Table 5. Manual measurements upon harvest.
It is important to schedule the measuring of plant growth. This ensures a steady stream of data over time. Especially when environmental conditions change (for example, the mean temperature), measurements need to have short intervals in between.
The vegetative phase of plant growth lasts until the first flower, at which point the reproductive phase starts. Measurement intervals should be shorter during reproductive growth, but this is no issue when growing basil. After all, basil is not grown for its flowers or fruits. Therefore, the measurement intervals can be kept at two to three times a week during the whole duration of the experiment.
For now, all data from the manual measurements needs to be collected in the Excel file 'Measurements Basil (user name)'. This is until a digital data collection point has been developed for the manual measurements. The Excel file can be downloaded below:
Be sure to always wash your hands before opening the kit to take measurements.
Two to three times a week, you need to measure:
- Branch number,
- Leaf number,
- Leaf area,
- Stem diameter,
- Stem length,
- pH *
- Electrical conductivity (EC) * * = If you have the appropriate sensors. Also write down the type and brand of these additional sensors in the manual measurements file.
Branch number: The number of branches of your plant does not include the stem itself. It also does not include the petioles of the leaves. The petiole is the small connection between the leaf and the stem or branch, as seen in the bottom left of figure 6.
Leaf number: When counting the leaf number, count all leaves that are more than 5 mm long. Do not count the two cotyledons (as indicated in figure 5 by the red arrows). Try not to touch the leaves too much during measurements, to reduce the chance of contamination. Also, plants are able to sense touch and might change their development if you touch them regularly in the same way.
Figure 5. The two cotyledons and two true leaves of sweet basil. The cotyledons (red arrows) are the first leaves that emerge from the seed embryo. They have a distinct shape, different from the true leaves (green arrows).
Leaf area: Measure the length and width of three developed leaves and enter the average length and width in the manual measurements file. The leaf area is difficult to measure by hand, and for bushy herbs such as basil also difficult to measure accurately by camera. Luckily, a good approximation can be made using the length and width of the leaves. If you enter these values in the excel sheet for manual measurement, a calculation for the leaf area is automatically made. When measuring the length and width, you measure the broadest part of the leaf for width. It is important that measure the length from the tip down to the base of the leaf, excluding the petiole. This is illustrated in figure 6 with two blue lines. (The petiole is the small connection between the leaf and the stem or branch, and is not considered part of the leaf. It should therefore not be measured for the leaf area calculation. In figure 6, it is marked with a red oval.)
Figure 6. Leaf area measurement of a true basil leaf. The red oval indicates the petiole, which should not be measured. Based on the blue length (L) and width (W), the calculation of basil leaf area (LA) is as follows: LA = 0,209 (L2 + W2) + 0,25 (With R2 = 0.895 and RMSE = 0.794) From: Modeling individual leaf area of basil (Ocimum basilicum) (Mousavi Bazaz et al., 2011).
Stem diameter: When measuring the stem, use a calliper for the diameter as this is more accurate that a ruler. Always measure the diameter at 3 cm above the base (the point where the stem ends and the root begins).
Before measuring the stem length, check for flowers and prune the basil if needed. (See 3A for instructions on pruning.)
Stem length: Measure the stem length from the base up to the highest point of the stem, the apex.
pH: Take the hydroponic kit out of the kit and remove the root compartment briefly. Measure the pH in the water of the nutrient compartment three times and write down the average as your measurement.
Electrical conductivity: Follow the same steps as described above for pH.
You should harvest your basil six weeks after it has germinated, according to the following steps:
- 1.Make the final regular manual measurements.
- 2.Disconnect your sensors or shut down the Astroplant kit entirely just before you harvest your plant. Otherwise redundant measurements on the empty kit will accumulate in the data files.
- 3.Take out the hydroponics system from the kit.
- 4.Disconnect the hydroponics and take the root compartment out of the larger nutrient reservoir.
- 5.Remove the irrigation ring and the top layer of clay pebbles. Be careful not to damage the roots; especially the small lateral roots are very vulnerable.
- 6.After removing as much of the top layer as possible, put the root compartment on its side.
- 7.Slide the rest of the clay pebbles together with the plant and its roots out of the compartment gently.
- 8.Cut the plant at its base, so that the root and shoot are separated. (In a hydroponic system, the base can be a little bit harder to pinpoint than for plants grown in soil. You can make the cut down where the stem is not green anymore, above any lateral roots.)
- 9.Now you are ready to measure the root length, root expanse, fresh weight and dry weight as explained below.
The measurements that are made after harvest are following the ‘New handbook for standardised measurement of plant functional traits worldwide’.[3]
- 1.Wash the separated root carefully using tap water to remove any dust or small particles. Only hold the root at the top near the cut, so you don't damage any lateral roots or root hairs.
- 2.Lay out the root on a flat and clean surface. Lay down the furthest root tips first, then the middle of the root system, towards the point where it was attached to the shoot. In this way you minimise the risk of damaging the root, while ensuring it is placed as stretched out as possible.
- 3.Use a regular ruler to measure the maximum length of the main root.
Measuring the development and expanse of the root system of the plant is very important to the development of the plant growth model. Often overlooked due to their inaccessibility, roots are critical in enabling the above-ground part of the plant to grow. Changes in their development are indicative for plant health, nutrient acquisition, water access, growth strategy and much more. Still, it is difficult and hugely time-consuming to measure the full root expanse. Therefore, the we will use software to extract data on the root system from scans or photographs. In order to obtain reliable data, it is important to make the best image possible. A scan is preferable, but high quality photographs are also adequate. In either case, follow these steps:
- 1.Lay the root out on a clean, clear and uniform background that contrasts clearly with the roots. A white plastic surface would be ideal.
- 2.Before making a scan or taking a picture, try to carefully arrange the roots so there is minimal overlap. This allows the software to better approximate the true expanse of the root system. It will be impossible to completely disentangle all the roots and you should not try to do this, as it will damage the root system.
- 3.Make several scans or pictures. When using a camera, take pictures from directly above the roots (90 degrees) and from slightly different angles (80 - 100 degrees).
The image analysis software is capable of dealing with a limited amount of root overlap, this is included in its calculation. Still, the data will be better if you were able to somewhat spread out the roots without damaging them.
For now, we ask you to send us the images of the root system for analysis (to [email protected]). There are currently several root phenotyping software packages that we are considering. Therefore, the results from the first experiments will be used to select a standard root analysis software, which may or may not be intuitively useable by the users themselves. You are of course encouraged to also experiment with these different software packages under consideration yourself. These are listed in the appendix (8-D).
For a detailed discussion of the various options in root phenotyping software, see Kuijken and colleagues (2015).[5]
Fresh weight:
- 1.Blot the plant parts dry with tissue paper to remove any surface water before measuring the fresh mass.
- 2.Use a scale with ±0,01 gram accuracy or better to weigh the root and shoot separately.
Dry weight:
- 1.Dry both the root and shoot at 70 °C for 72 hours.
- 2.Weigh the plant parts immediately after taking them out of the oven, otherwise the samples will take up some moisture from the air and become heavier. (If you don’t weigh them immediately, put them in a closed container with silica gel until weighing.)
- 3.Use a scale with ±0.001 gram accuracy or better to weigh the root and shoot separately.
- 1.Wash the clay pebbles at least three times thoroughly to rinse off residue and dust.
- 2.If you have access to a pH sensor, it is recommended to check the pH of the pebbles: Put some pebbles in a glass of demineralised water (pH<4.0). Stir well and leave to rest. Measure the pH the next day. If it is above pH 7.0, then soak overnight in an acidic solution (pH<4.0). Then rinse thoroughly five times over.
- 3.Sterilise with 20% commercial bleach or hydrogen peroxide solution. Use the same cleaning product for all containers, tubing and the pump.
- 4.Rinse all cleaned parts of the kit with tap water five times after using the cleaning product.
If you think you have found a fungal or bacterial infection on your plant, go to the appendix (9-B) to see if you can confirm and identify the infection. If it is indeed an (minor) infection, the experiment can still continue if you follow these steps: Firstly, document where the infection is: on only one leaf, on multiple leaves, on the stem, or on the roots. Take note of the infection and its progress in your manual measurement file. Secondly, try to contain the infection so you can continue the experiment. If it is an infection on one or more leaves, those leaves need to be removed from the plant. Thirdly, the inside of the kit needs to be cleaned with a standard cleaning product. Make sure that you screen the plant often, preferably daily, for the next two weeks to see if the infection has been contained or is still spreading further. When you find additional infected leaves, remove them as well. Do not forget to document this as well. At the end of the experiment, even if the infection was easily contained, the entire kit needs to be cleaned carefully.
If your plant has lost half of its leaves or more to the infection, it cannot be saved anymore. The best option is then to remove the plant and thoroughly clean the AstroPlant kit (especially the hydroponic system!) before starting a new experiment. Otherwise the pathogen or its spores will remain inside the kit and contaminate all future experiments.
Although the AstroPlant Growth Protocol aims to be all-encompassing, some issues might not be addressed yet. In case you have any questions about your experiment or the AstroPlant project, you can always contact AstroPlant for support. A centralized support system is being developed at the moment, which will allow questions or remarks to be redirected to the right person within the AstroPlant team automatically.
One general remark on plant related problems: In principle the experiment is meant to investigate plant growth under various conditions, some good, some less so. A plant not growing as quickly as expected or dying off might be the result of environmental conditions that are being recorded. In that case it is not a failed experiment, just one of the possible outcomes of the experiment. In fact, these results are crucial to the goal of understanding plant growth and development.
For basil growth, use a general 7-4-10 NPK fertiliser solution with micronutrients. This composition is good for general vegetative phase plant growth.
The entire nutrient solution needs to be chanced once every 14 days (see 8C for details). Fill the nutrient solution reservoir with 6 litres of fertiliser solution, prepared as proscribed on the bottle.
The growth substrate of choice is a type of small clay pebble called LECA. These clay pebbles are available everywhere and reusable for all experiments. Examples of suitable LECA products include 'Grorox' from GHE and 'Euro Pebbles' from Plagron (Sometimes they are called differently, like 'hydropebbles', depending on the company.) Acquiring 5 litres of LECA is enough to fill the root compartment. The clay pebbles should not be placed in the nutrient solution reservoir.
The clay pebbles in the root compartment allow the nutrient solution to flow over the roots back into the reservoir at the bottom of the bucket. These pebbles don’t react with the nutrients and don’t hold much water in comparison to other hydroponic media. Roots that grow in between the pebbles therefore have access to a continuous small flow of nutrients, ensuring good nutrition and an oxygen rich environment at the same time.
Table 6. Properties of the AstroPlant LEDs. The AstroPlant top panel contains 4 blue, 8 red and 4 far red LEDs. In the recommended settings of the Growth Protocol this 1:2:1 ratio has been accounted for.
If you choose to conduct an experiment with one of the LED light groups completely off, you need to set the value to '0.0', instead of 'disabled' during the setup. Any changes also need to take into account the 1:2:1 (B:R:FR) ratio of the number of LEDs on the AstroPlant top panel.
For more details on other light combinations and their effect on basil growth and nutrient content, see: Artificial LED lighting enhances growth characteristics and total phenolic content of Ocimum basilicum, but variably affects transplant success (Bantis et al., 2016) Light-emitting diodes; on the way to combinatorial lighting technologies for basic research and crop production (Tarakanov et al., 2012) Effect of supplemental UV-A irradiation in solid-state lighting on the growth and phytochemical content of microgreens (Brazaityte et al., 2015)
The nutrient solution in the kit needs to be fully replaced every two weeks. This ensures that the plants in the experiment have good and stable access to nutrients over the course of the experiment.
As the plant takes up nutrients in a hydroponic system, the concentration of nutrients in the solution depletes. Less nutrition will be available for the plant and the balance of the solution will also change, as can be seen in table 7. Replacing the entire solution of the kit every two weeks prevents the values from diverging too much from the experimental standard.
Table 7. Nutrient depletion for basil in a hydroponic system with 2 EC starting value (2,6-1-2 NPK). Changes in the nutrient content and balance of a hydroponic nutrients solution for basil growing under high DLI. From: Effects of Nutrient Concentration and Daily Light Integral on Growth and Nutrient Concentration of Basil Species in Hydroponic Production (Walters & Currey, 2018)
[ Under construction ]
The following root phenotyping software packages currently under consideration:
SmartRoot Lobet, G., Pagès, L., & Draye, X. (2011). A novel image analysis toolbox enabling quantitative analysis of root system architecture. Plant physiology, 157, pp 29-39.
RootReader Clark, R. T., Famoso, A. N., Zhao, K., Shaff, J. E., Craft, E. J., Bustamante, C. D., ... & Kochian, L. V. (2013). High‐throughput two‐dimensional root system phenotyping platform facilitates genetic analysis of root growth and development. Plant, cell & environment, 36(2), 454-466.
DIRT Das, A., Schneider, H., Burridge, J., Ascanio, A. K. M., Wojciechowski, T., Topp, C. N., ... & Bucksch, A. (2015). Digital imaging of root traits (DIRT): a high-throughput computing and collaboration platform for field-based root phenomics. Plant methods, 11(1), 51.
GiA Roots Galkovskyi, T., Mileyko, Y., Bucksch, A., Moore, B., Symonova, O., Price, C. A., ... & Harer, J. (2012). GiA Roots: software for the high throughput analysis of plant root system architecture. BMC plant biology, 12(1), 116.
[1] Pushpangadan, P., & George, V. (2012). Basil. In Handbook of Herbs and Spices (Second Edition), Volume 1 (pp. 55-72). [2] Frąszczak, B., Golcz, A., Zawirska-Wojtasiak, R., & Janowska, B. (2014). Growth rate of sweet basil and lemon balm plants grown under fluorescent lamps and LED modules. Acta Sci. Pol. Hortorum Cultus, 13(2), 3-13. [3] Peìrez-Harguindeguy, N., Díaz, S., Garnier, E., Lavorel, S., Poorter, H., Jaureguiberry, P., Bret-Harte, M. S., Cornwell, W. K., Craine, J. M., Gurvich, D. E., Urcelay, C, Veneklaas, E. J., Reich, P. B., Poorter, L., Wright, I. J., Ray, P., Enrico, L., Pausas, J. G., De Vos, A. C., Buchmann, N., Funes, G., Quétier, F., Hodgson, J. G., Thompson, K., Morgan, H. D., Ter Steege, H., Sack, L., Blonder, B.,Poschlod, P., Vaieretti, M. V., Conti, G., Staver, A. C., Aquino, S. & Cornelissen, J. H. C.(2016). New handbook for standardised measurement of plant functional traits worldwide. Aust. J. Bot, 64, 715–716. [4] Kuijken, R. C., Van Eeuwijk, F. A., Marcelis, L. F., & Bouwmeester, H. J. (2015). Root phenotyping: from component trait in the lab to breeding. Journal of experimental botany, 66(18), 5389-5401. Figure 1 from: Carvalho, S. D., Schwieterman, M. L., Abrahan, C. E., Colquhoun, T. A., & Folta, K. M. (2016). Light quality dependent changes in morphology, antioxidant capacity, and volatile production in sweet basil (Ocimum basilicum). Frontiers in plant science, 7, 1328.