An improved method for the segmentation of roots from X-ray computed tomography 3D images: Rootine v.2

Background X-ray computed tomography is acknowledged as a powerful tool for the study of root system architecture of plants growing in soil. In this paper, we improved the original root segmentation algorithm “Rootine” and present its succeeding version “Rootine v.2”. In addition to gray value information, Rootine algorithms are based on shape detection of cylindrical roots. Both algorithms are macros for the ImageJ software and are made freely available to the public. New features in Rootine v.2 are (i) a pot wall detection and removal step to avoid segmentation artefacts for roots growing along the pot wall, (ii) a calculation of the root average gray value based on a histogram analysis, (iii) an automatic calculation of thresholds for hysteresis thresholding of the tubeness image to reduce the number of parameters and (iv) a false negatives recovery based on shape criteria to increase root recovery. We compare the segmentation results of Rootine v.1 and Rootine v.2 with the results of root washing and subsequent analysis with WinRhizo. We use a benchmark dataset of maize roots (Zea mays L. cv. B73) grown in repacked soil for two scenarios with differing soil heterogeneity and image quality. Results We demonstrate that Rootine v.2 outperforms its preceding version in terms of root recovery and enables to match better the root diameter distribution data obtained with root washing. Despite a longer processing time, Rootine v.2 comprises less user-defined parameters and shows an overall greater usability. Conclusion The proposed method facilitates higher root detection accuracy than its predecessor and has the potential for improving high-throughput root phenotyping procedures based on X-ray computed tomography data analysis.

An Improved Method for the Segmentation of Roots from X-ray Computed Tomography 3D Images: Rootine v.2

BackgroundX-ray computed tomography is acknowledged as a powerful tool for the study of root system architecture of plants growing in soil. In this paper, we improved the original root segmentation algorithm “Rootine” and present its succeeding version “Rootine v.2”. In addition to grey value information, Rootine algorithms are based on shape detection of cylindrical roots. Both algorithms are macros for the ImageJ software and are made freely available to the public. New features in Rootine v.2 are (1) a pot wall detection and removal step to avoid segmentation artefacts for roots growing along the pot wall, (2) a calculation of the root average grey value based on a histogram analysis, (3) an automatic calculation of thresholds for hysteresis thresholding of the tubeness image to reduce the number of parameters and (4) a false negatives recovery based on shape criteria to increase root recovery. We compare the segmentation results of Rootine v.1 and Rootine v.2 with the results of root washing and subsequent analysis with WinRhizo. We use a benchmark dataset of maize roots (Zea mays L. cv. B73) grown in repacked soil for two scenarios with differing soil heterogeneity and image quality. ResultsWe demonstrate that Rootine v.2 outperforms its preceding version in terms of root recovery and enables to match better the root diameter distribution data obtained with root washing. Despite a longer processing time, Rootine v.2 comprises less user-defined parameters and shows an overall greater usability. ConclusionThe proposed method facilitates higher root detection accuracy than its predecessor and has the potential for improving high-throughput root phenotyping procedures based on X-ray CT data analysis.

Wheat Varietal Differences in Below Ground Biomass Revealed by a Semi-Quantitative Estimation of Wheat Root DNA in Soil Samples

Root research on field grown crops is hindered by the difficulty of estimating root biomass in soil. Root washing, the current standard method is laborious and expensive. Biochemical methods to quantify root biomass in soil, targeting species-specific DNA, have potential as a more efficient assay. We combined an efficient DNA extraction method, designed specifically to extract DNA from soil, with well-established quantitative PCR methods to estimate the root biomass of twenty-two wheat varieties grown in field trials over two seasons. We also developed an assay for estimating root biomass for black-grass, a common weed of wheat cultivation. Two robust qPCR assays were developed to estimate the quantity of plant root DNA in soil samples, one specific to wheat and barley, and a second specific to black-grass. The DNA qPCR method was comparable, with high correlations, with the results of root washing from soil cores taken from winter wheat field trials. The DNA qPCR assay showed both variety and depth as significant factors in the distribution of root biomass in replicated field trials. The results suggest that these DNA qPCR assays are a useful, high throughput tool for investigating the genetic basis of wheat root biomass distribution in field grown crops, and the impact of black-grass root systems on crop production.

Studies on certain aspects of root surface fungi II. Succession of fungi on decomposing Pennisetum typhoides (Burm. f.) Stapf et Hubb.

Sixty seven fungal species from the nonrhizosphere (NR), rhizopshere (RS) and cortical (RPC) and steler (RPS) parts of rhizopshere of crown (RC), middle (RM) and distal (RD) regions of decomposing roots of <i>Pennisetum typhoides</i> Burm. f.) Stapf. et Hubb. were isolated during December to June, 1970-72. The number of fungal species gradually decreased from NR—RPS in horizontal and RC—RD in vertical regions. The fungal population was always higher in RS of different depths than in corresponding NR regions. The amino acids and sugar components of the roots showed a direct correlation with the fungal population. The amount of cellulose, hemicellulose and lignin components of roots gradually decreased from December to June. Root-washing collected from RC, RM and RD regions exhibited the presence of vanillic acid and 3-4 dihydroxy benzoic acid during March and April. It also exerted an adverse effect on the 10 rhizosphere fungi during this period. pH and moisture contents showed a poor correlation with the fungal population cxcept during summer months. <i>Phycomycetes</i> with species of <i>Deuteromycetes</i> obtained in the first phase were followed by<i> Deuteromycetes</i> along with few <i>Ascomycetes</i> in the second phase. In the last Deuteromycetes with some slerile mycelia were isolated. Aspergilli were the most numerous throughout the present investigation.

An Automatic Aeroponics Growth System for Bamboo Seedling and Root Observation

According to the biological characteristics of bamboo seedling, an automatic aeroponics growth system is developed for bamboo seedling and root observation, which can prepare good condition of water-fertilizer, air and warm during bamboo seedling. The ultrasonic atomizer is used to atomize the nutrient solution to the ultrafine particles of 1-5 microns diameter. Compared with traditional piezometrical atomization, this method can not only improve atomization efficiency of nutrient solution to promote uniform absorption at the roots, but also avoid the phenomenon of root-washing. In addition, considering the significant effect of temperature and humidity on bamboo root growth, a temperature-humidity control system is designed for automatic control of water-fertilizer and temperature in bamboo root growth environment. The system supplies an experimental platform with features of simple structure and convenient control. In the procedure of bamboo seedling, bamboo rhizome and shoot can grow fast because of enough moisture nutrition, good breathing, and low growth resistance. Furthermore, it is also convenient for morphologic observation of bamboo roots.

Aboveground Screening for Genotypic Differences in Cucumber Root Growth in the Greenhouse and Field

A simple, aboveground method to study cucumber (Cucumis sativus L.) root growth was developed using a subsurface herbicide banding technique. Those plants with roots that grow deeper or faster reach the herbicide sooner and exhibit herbicide injury symptoms sooner. Greenhouse pot trials showed that 0.25 or 0.50 kg simazine/ha could be used to produce distinctive symptoms; time to symptom expression increased with the depth of the band from the soil surface. Root washing experiments verified that root length was associated with response time. In field trials, response time and severity of symptoms varied with herbicide concentration, depth, and distance from the seed row, thereby providing an indication of where the roots were in the soil. About 100 diverse cucumber genotypes were tested for differences in root growth rate in the greenhouse and in the field. Time to symptom expression was normally distributed among the genotypes; analysis of variance (ANOVA) indicated significant genotypic differences. This system can be used for cultural or physiological studies, or nondestructively for selection and breeding purposes. If the herbicide is placed sufficiently deep to prevent damage to the cotyledons, the plants are capable of flowering and producing fruit. Chemical name used: 6-chloro-N, N′-diethyl-1,3,5-triazine-2,4-diamine (simazine).

An efficient method to evaluate alfalfa cultivars for resistance to verticillium wilt

A growth-room technique was developed to evaluate alfalfa cultivars for resistance to verticillium wilt caused by Verticillium alba-atrum Reinke & Berthold. The process involves growing alfalfa seedlings in Cornell mix in Roottrainers™ and inoculating the seedlings in batches of 96, without digging, root-washing or transplanting individual seedlings. This method was shown to be effective and efficient in screening large numbers of alfalfa seedlings, approximately 1000 seedlings m−2, using little growth-room space and minimum labor. Key words: Verticillium albo-atrum, Medicago sativa, resistance, technique, alfalfa

ABOVE-GROUND SCREENING FOR GENETIC VARIATION IN CUCUMBER ROOT GROWTH

An above ground screening method to study cucumber root growth was developed using the herb icicle banding technique of Robertson et al. (Crop Sci 25:1084, 1985). Those roots that grow deeper or faster, sooner reach the herbicide, and sooner exhibit herbicide damage symptoms. Greenhouse pot trials showed that 1/4-1/2 lb/A atrazine could be used to produce distinctive symptoms, differentiate between depths of banding, and among different genotypes. Based on root washing experiments of a few cultivars, root length and/or mass correlated with herbicide symptom expression. One hundred diverse cucumber genotypes were tested in the greenhouse. Time to symptom expression was normally distributed among the genotypes; analysis of variance indicated significant genotypic differences. The herbicide banding technique was also useful for monitoring cucumber root growth in the field. Response time and severity varied with herbicide concentration, depth, and distance from the seed row. The diverse cucumber genotypes are now being tested in the field to further determine if there are significant genotypic differences and to compare greenhouse and field behavior.

Portable apparatus for separating roots from soil cores

One of the most widely used methods of root study involves obtaining soil cores and washing the roots contained free of soil (Wellbank & Williams, 1968; Ellis & Barnes, 1971; Kolesnikov, 1971; Schuurman & Geodewaagen, 1971; Bohm, Maduakor & Taylor, 1977). However, the process of root washing is time consuming and laborious. Some of the washing assemblies elsewhere (Peyev, 1952; Schuurman & Goedewaagen, 1971; Wellbank et al. 1974) have not gained popularity with researchers in developing countries because of the high costs involved or the sophisticated facilities needed. There is therefore a need for a low-cost root washing apparatus which can be fabricated with locally available materials. This note reports the development of such an apparatus.