Monitoring of plant development in controlled environment with machine vision.

Information acquisition is the foremost requirement for the control and continued operation of any complex system. This is especially true when a plant production system is used as a major component in a sustainable life support system. The plant production system not only provides food and fiber but is a means of providing critically needed life supporting elements such as O2 and purified H2O. The success of the plant production system relies on close monitoring and control of the production system. Machine vision technology was evaluated for the monitoring of plant health and development and showed promising results. Spectral and morphological characteristics of a model plant were studied under various artificially induced stress conditions. From the spectroscopic studies, it was found that the stresses can be determined from visual and non-visual symptoms. The development of the plant can also be quantified using a video image analysis base approach. The correlations between the qualities of the model plant and machine vision measured spectral features were established. The success of the research has shown a great potential in building an automated, closed-loop plant production system in controlled environments.     

Instrumentation for plant health and growth.

Comprehensive spectroscopic monitoring of plant health and growth in bioregenerative life support system environments is possible using a variety of spectrometric technologies. Absorption spectrometry and atomic emission spectrometry in combination allow for direct, on-line, reagentless monitoring of plant nutrients from nitrate and potassium to micronutrients such as copper and zinc. Fluorometric spectrometry is ideal for the on-line detection, identification and quantification of bacteria and fungi. Liquid Atomic Emission Spectrometry (LAES) is a new form of spectrometry that allows for direct measurement of atomic emission spectra in liquids. An electric arc is generated by a pair of electrodes in the liquid to provide the energy necessary to break molecular bonds and reduce the substance to atomic form. With a fiber probe attached to the electrodes, spectral light can be transmitted to a photodiode array spectrometer for light dispersion and analysis. Ultraviolet (UV) absorption spectrometry is a long-established technology, but applications typically have required specific reagents to produce an analyte-specific absorption. Nitrate and iron nutrients have native UV absorption spectra that have been used to accurately determine nutrient concentrations at the +/- 5% level. Fluorescence detection and characterization of microbes is based upon the native fluorescent signatures of most microbiological species. Spectral and time-resolved fluorometers operating with remote fiber-optic probes will be used for on-line microbial monitoring in plant nutrient streams.     

Optical sensors for monitoring and control of plant growth systems.

Optical chemical sensors have been developed for monitoring several parameters relevant to plant growth systems. These sensors utilize porous polymer and porous glass as the sensing element, and optical fiber input/output lines connected to a custom optoelectronic interface. Present in the sensing element are immobilized colorimetric indicators, which react with the analyte to be sensed. This reaction results in a change in the optical properties of the sensor. These sensors are particularly suited to in-situ monitoring of nutrient solution parameters and atmospheric trace contaminants in life support and plant growth systems. Sensors for monitoring pH, ammonia, and ethylene will be discussed.     

Analysis of remote reflection spectroscopy to monitor plant health.

Remote non-contact reflection spectroscopy is examined as a method for detecting stress in Controlled Ecological Life Support System CELSS type crops. Lettuce (Lactuca [correction of Latuca] Sativa L. cv. Waldmans Green) and wheat (Triticum Aestivum L. cv. Yecora Rojo) were grown hydroponically. Copper and zinc treatments provided toxic conditions. Nitrogen, phosphorous, and potassium treatments were used for deficiency conditions. Water stress was also induced in test plants. Reflectance spectra were obtained in the visible and near infrared (400nm to 2600nm) wavebands. Numerous effects of stress conditions can be observed in the collected spectra and this technique appears to have promise as a remote monitor of plant health, but significant research remains to be conducted to realize the promise.     

Novel sensor technology for monitoring and control of critical plant nutrient parameters.

A novel dielectric sensor technology has been developed for monitoring and control of plant nutrient delivery systems as part of NASA's Controlled Ecological Life Support System (CELSS) program. A unique measurement phenomenon was discovered in which the electrostatic field is shunted to a third terminal of the sensor, resulting in a much greater sensitivity to changes in the complex dielectric properties of the nutrient solution. Based on this phenomenon, a small, flexible, thin-film shunting dielectric sensor (SDS) was designed to provide low-frequency, non-invasive measurement of both the thickness and nutrient concentration of the layer of solution on a plant growth surface. Test results indicate a sensitivity of +/- 0.05mm in layer thickness while characterization of the ability to measure nutrient concentration continues. The development plan for this sensor is presented and other applications are discussed.     

Status and progress in on-line spectrometric monitoring and control of plant nutrient solutions.

Biotronics has been involved, under NASA sponsorship, in a wide ranging research and development program for instrumentation used in the monitoring and control of controlled environment agriculture. This program has embraced both chemical monitoring of plant nutrient solutions as well as microbiological monitoring of bacteria and fungi in these same solutions. This paper emphasizes the microbiological monitoring aspects of this program. In contrast to traditional methods of microbiological analysis based on culturing, staining and microscopic observation, the development described here is based on spectroscopic measurements, more specifically spectral fluorometry. The rationale, objectives, analytical methods and new instrumentation employed in the development of an on-line microbiological analyzer (MBA) are presented in some detail. Finally, the signal processing/pattern recognition methods used to evaluate the spectral data and produce estimates of microbial populations are described along with experimental test results to conclude the paper.     

Force sensitivity of plant gravisensing.

Rotation at 4, 10, 50 and 100 rpm on a horizontal clinostat and in microgravity exerts limited effects on the morphogenesis of lettuce and cress root statocytes and statoliths if compared with the vertical control or 1 g spaceflight reference centrifuge. However, the average distance of statoliths from the distal wall increases. The pattern of plastid location of microgravity-grown and that of clino-rotated samples has been determined at 10, 50, and 100 rpm. Experiments on the centrifuge-clinostat and spaceflight centrifuge (acceleration forces of 0.005 to 1 g) revealed that the average statolith location depends on the amplitude of acropetally or basipetally directed mass acceleration. Decreasing the acropetally directed force from 1 g to 0.4 g dislocates statoliths towards the cell center possibly mediated by the elastic forces of the cytoskeleton. In statocytes formed on the clinostat or in microgravity, the majority of statoliths are located at the center of the cell. To force the statoliths from the center of the statocyte towards one of its poles, a threshold mass acceleration of 0.01 g is required. Statocytes with centrally-located statoliths are considerably more effective in transducing a gravistimulus than those with distally-located plastids. The latent time of the graviresponse is shorter and the response itself is enhanced in roots grown on the clinostat compared to vertically grown samples. The early phases of graviperception are independent of root growth conditions since presentation time and g-threshold are similar for roots grown stationary and those on a clinostat. We propose a sequence of events in gravitropic stimulation that considers not only the lateral displacement of statoliths, as predicted by the starch-statolith hypothesis, but also its longitudinal motion, together with differential gravisensitivity of mechanotransducing structures along the lower-most longitudinal cell wall.     

Oscillations of axial plant organs.

The tips of roots and shoots commonly show lateral movements as they grow forwards. These occur as both circumnutations (with long periods and large amplitudes) and micronutations (with short periods and small amplitudes). Their properties are reviewed, with emphasis on roots, and possible ways in which they could be regulated are discussed. The mechanisms could include long-range controls (for circumnutations) that depend on transmissible signals using steps common to gravitropism, and short-range controls (for micronutations) that operate within the elongation zone. The former are a property of the apex as a whole, while the latter may be confined to localized groups of cells. Simulation of nutations is presented with a view to isolating key physiological processes. However, this approach is limited by the current inadequate understanding of the growth mechanisms involved.     

Theoretical considerations of plant gravisensing.

The mechanisms proposed to explain gravity sensing can be divided into two groups, "statolith" and "non-statolith" mechanisms. The traditional estimates of the plausibility of these mechanisms are based on the analysis of the signal-to-noise ratio. The existing data indicate that the problem of plant gravisensing may be related to the general problem of the detection of weak signals in mechanoreceptors. This paper reviews the known mechanisms of plant gravisensing as well as the latest nonlinear stochastic models of mechanoreception in which noise promotes detection and amplification of weak signals. These models based on nonlinear stochastic phenomena may be used to explain plant gravisensing, if the cell is considered a dynamic, spatially distributed system of active intracellular cytoskeletal networks and mechanosensitive proteins.     

Planetarium Inversum -- a space vision for Earth education.

In a planetarium, the visitor is sitting on Earth and looking into an imaginary space. The Planetarium Inversum is the opposite: visitors are sitting in a space station, looking down on Mother Earth. It is a scientifically-based information show with visitors involvement, its elements being partially virtual (Earth in space has to be projected with highest possible resolution) but also containing real structures, such as the visitors' Earth observatory with adjacent biological systems (plant cultures and other ecological life support components). Its main message concerns the limits and the vulnerability of our home planet, its uniqueness, beauty and above all, its irreplaceableness: Earth does not have an emergency exit. The Earth observatory is part of a ring shaped, rotating space station of the type designed by Wernher von Braun decades ago. Visitors are told that gravity is being substituted by centrifugal force. Both types of life support systems are being demonstrated--self regenerative life based ones and technical ones as a backup (solar electric splitting of water and chemical absorption of respiratory CO2).     

Cell biology of plant gravity sensing.

The debate about whether gravity sensing relies upon statoliths (amyloplasts that sediment) has intensified with recent findings of gravitropism in starchless mutants and of claims of hydrostatic gravity sensing. Starch and significant plastid sedimentation are not necessary for reduced sensing in mutant roots, but plastids might function here if there were a specialized receptor for plastid mass e.g. in the ER. Alternatively, components in addition to amyloplasts might provide mass for sensing. The nucleus is dense and its position is regulated, but no direct data exist for its role in sensing. If the weight of the protoplast functioned in sensing, why would there be specific cytological specializations favoring sedimentation rather than cell mass? Gravity has multiple effects on plants in addition to gravitropism. There may be more than one mechanism of gravity sensing.     

Effects of air current speed on gas exchange in plant leaves and plant canopies.

To obtain basic data on adequate air circulation to enhance plant growth in a closed plant culture system in a controlled ecological life support system (CELSS), an investigation was made of the effects of the air current speed ranging from 0.01 to 1.0 m s-1 on photosynthesis and transpiration in sweetpotato leaves and photosynthesis in tomato seedlings canopies. The gas exchange rates in leaves and canopies were determined by using a chamber method with an infrared gas analyzer. The net photosynthetic rate and the transpiration rate increased significantly as the air current speeds increased from 0.01 to 0.2 m s-1. The transpiration rate increased gradually at air current speeds ranging from 0.2 to 1.0 m s-1 while the net photosynthetic rate was almost constant at air current speeds ranging from 0.5 to 1.0 m s-1. The increase in the net photosynthetic and transpiration rates were strongly dependent on decreased boundary-layer resistances against gas diffusion. The net photosynthetic rate of the plant canopy was doubled by an increased air current speed from 0.1 to 1.0 m s-1 above the plant canopy. The results demonstrate the importance of air movement around plants for enhancing the gas exchange in the leaf, especially in plant canopies in the CELSS.     

The effect of gravity on plant germination.

An axis clinostat was constructed to create micro and negative gravity also a rotated flat disk was constructed with different rotation rates to give increased gravity, by centrifugal force up to 48 g. Rice seeds were grown on agar in tubes at the constant air temperature of 20 degrees C under an average light condition of 110 micromol/m2/sec(PPF). Humidity was not controlled but was maintained above 90%. Since the tube containers were not large enough for long cultivation, shoot and root growth were observed every 12 hours until the sixth day from seeding. The lengths of shoots and roots for each individual plant were measured on the last day. The stem lengths were increased by microgravity but the root lengths were not. Under the negative gravity, negative orthogeotropism and under microgravity, diageotropism was observed. No significant effect of increased gravity was observed on shoot and root growth.     

Spectral composition of light and plant productivity.

Among other problems the Institute of Biophysics is working on the development of physiological and fundamental aspects of intensive light cultivation of higher plants. These technologies can be used in life support systems for stationary space station such as a Lunar base, a planetary base or a large orbital station. The source of energy may be the Sun or a nuclear reactor. In certain conditions, such sources of energy allow the use of a very broad range of irradiance of plants, in particular in the light energy range up to 2-3 times the solar energy (up to 100-1200 W/m2 PAR). Our Institute was the first to show that under such a high irradiance, some plants (radish, wheat, for example) can actively photosynthesize and exhibit high productivity on a sowing area basis. These results were later confirmed in the laboratory of Prof. Salisbury (USA).     

The function of calcium in plant graviperception.

The fundamental question of gravitational biology is how do plants perceive a gravity. Recent experimental results have demonstrated that Ca second-messenger system has an essential role in induction of graviresponsiveness. Our data, that stimuli of various nature cause a rise of hyaloplasm Ca level revealed by means of pyroantimonate method, as well as complete inhibition of the gravitropism in roots of pea seedlings, provide indirect but consistent evidence of this role of Ca ions. A possible explanation for these results is that they may be due to an unbalanced and undirectional influx of Ca ions in statocytes from cell walls or from intracellular Ca stores, while in the presence of the Earths 1 g vector, this process occurs directionally, along this vector. It is possible that a target for the gravity stimulus is the flux mechanism of Ca to statocytes, including participation of the phosphatidylinositol system and calmodulin. The data that have become available from space flight experiments will be reviewed and an attempt will be made to compare these results with ground-based observations.     

Progress in plant research in space.

Progress is reviewed of spaceflight research conducted with plants between 1987 and 1992. Numerous plant experiments have been performed on spacecraft and sounding rockets in the past five years by scientists of the US, the former Soviet Union, Europe, and other areas. The experiments are categorized into three areas: gravity sensing, transduction, and response; development and reproduction; and metabolism, photosynthesis, and transport. The results of these experiments continue to demonstrate that gravity and/or other factors of spaceflight affect plants at the organismal, cellular, subcellular, and molecular levels, resulting in changes in orientation, development, metabolism, and growth. The challenge now is to truly dissect the effects of gravity from those of other spaceflight factors and to identify the basic mechanisms underlying gravity's effects.     

Microcomputer-based monitoring of cardiovascular functions in simulated microgravity.

A microcomputer-based system for non-invasive monitoring of cardiovascular system in simulated microgravity is described. The system evaluates automatically, accurately and interactively heart beat intervals, beat-to-beat non-invasive finger arterial blood pressure (systolic, diastolic, mean and pulse pressure) using a Finapres device and beat-to-beat changes of thoracic blood volume using impedance changes. In addition, beat-to-beat evaluation of cardiac mechanical function including left ventricular ejection time, diastolic time, systolic time intervals, left ventricular ejection fraction estimate and several other contractility parameters, left ventricular volume, stroke volume and cardiac output estimates are performed with high degree of automaticity.     

Radiosensibility of higher plant seeds after space flight.

The influence of long-term storage of higher plant seeds under space flight conditions (49 to 827 days) on their radiosensibility was studied in the experiments on the orbital stations Salyut 6 and 7. Short-term storage has been proved to have no effect on radiosensitivity of Crepis capillaris seeds. Only in the case of maximal exposure duration the frequency of chromosome aberrations in post-flight irradiated seeds significantly exceeded the chromosome aberration frequency in the ground-based irradiated control. A statistically significant increase in the number of cells with multiple chromosome aberrations was also observed in this experiment. After gamma-irradiation of Arabidopsis thaliana seeds the germinating ability and survival rate of plants decreased depending on the duration of seed storage. Flight-exposed seeds were more sensitive to irradiations with respect to these parameters. A statistically significant increase in the frequency of recessive lethal mutations was observed only in two experiments of long exposure duration.     

Fundamental study on gas monitoring in CELSS.

A mass spectrometer and computer system was developed for conducting a fundamental study on gas monitoring in CELSS. Respiration and metabolism of the hamster and photosynthesis of the Spirulina were measured in a combination system consisting of a hamster chamber and a Spirulina cultivator. They are connected through a membrane gas exchanger. Some technical problems were examined. In the mass spectrometric gas monitoring, a simultaneous multi-sample measurement was developed by employing a rotating exchange valve. Long term precise measurement was obtained by employing an automatic calibration system. The membrane gas sampling probe proved to be useful for long term measurement. The cultivation rate of the Spirulina was effectively changed by controlling CO2 and light supply. The experimental results are helpful for improving the hamster-spirulina system.     

Gravitational effects on plant growth hormone concentration.

Numerous studies, particularly those of H. Dolk in the 1930's, established by means of bio-assay, that more growth hormone diffused from the lower, than from the upper side of a gravity-stimulated plant shoot. Now, using an isotope dilution assay, with 4,5,6,7 tetradeutero indole-3-acetic acid as internal standard, and selected ion monitoring-gas chromatography-mass spectrometry as the method of determination, we have confirmed Dolk's finding and established that the asymmetrically distributed hormone is, in fact, indole-3-acetic acid (IAA). This is the first physico-chemical demonstration that there is more free IAA on the lower sides of a geo-stimulated plant shoot. We have also shown that free IAA occurs primarily in the conductive vascular tissues of the shoot, whereas IAA esters predominate in the growing cortical cells. Now, using an especially sensitive gas chromatographic isotope dilution assay we have found that the hormone asymmetry also occurs in the non-vascular tissue. Currently, efforts are directed to developing isotope dilution assays, with picogram sensitivity, to determine how this asymmetry of IAA distribution is attained so as to better understand how the plant perceives the geo-stimulus.