Agronomy, Volume 8, Issue 5 (May 2018) – 21 articles

Precision agriculture (PA) has been used for ≥25 years to optimize inputs, maximize profit, and minimize negative environmental impacts. Legumes play an important role in cropping systems, by associating with rhizobia microbes that convert plant-unavailable atmospheric nitrogen into usable nitrogen through symbiotic nitrogen fixation (SNF). However, there can be field-level spatial variability for SNF activity, as well as underlying soil factors that influence SNF (e.g., macro/micronutrients, pH, and rhizobia). There is a need for PA tools that can diagnose spatial variability in SNF activity, as well as the relevant environmental factors that influence SNF. Little information is available in the literature concerning the potential of PA to diagnose/optimize SNF. Here, we critically analyze SNF/soil diagnostic methods that hold promise as PA tools in the short–medium term. We also review the challenges facing additional diagnostics currently used for research, and describe the innovations needed to move them forward as PA tools. Our analysis suggests that the nitrogen difference method, isotope methods, and proximal and remote sensing techniques hold promise for diagnosing field-level variability in SNF. With respect to soil diagnostics, soil sensors and remote sensing techniques for nitrogen, phosphorus, pH, and salinity have short–medium term potential to optimize legume SNF under field conditions. Full article

An efficient callus induction and in vitro regeneration were developed using plant growth regulators, carbon sources, and basal media for three selected Malaysian wetland rice varieties (MR220, MR220-CL2, and MR232) and one upland variety (Bario). Effect of plant growth regulator (PGR) was carried out using four different concentrations (1–4 mg/L) of 2,4-D (2, 4-dichlorophenoxyacetic acid), and NAA (1-naphthalene acetic acid) (2.5, 5.0, 7.5, and 10 mg/L) with optimized 2,4-D. Effects of carbon sources (maltose and sorbitol), and basal media (MS, N6, and LS) were also studied with optimized PGR to maximize the induction of regenerable calli. This study found that all four varieties exhibited high frequency of callus induction on MS (Murashige and Skoog) medium that was supplemented with 3 mg/L 2,4-D and 30 g/L maltose. Callus induction frequencies in the cases of MR220, MR220-CL2, MR232, and Bario were found to be 76%, 94%, 85%, and 42% respectively. Morphological analysis through scanning electron microscopy (SEM) and histological analysis revealed the embryogenicity of the induced callus. In the regeneration study, it was observed that combination of 2 mg/L BAP (6-benzylaminopurine), 2 mg/L Kin (Kinetin) and 0.5 mg/L NAA supplemented MS medium has the potential to promote regeneration of selected indica rice varieties with higher regeneration percentage, i.e., 82% (MR220-CL2), 68% (both in MR220 and MR232), and 40% (Bario). The optimized conditions for callus formation and regeneration can be useful for biotechnological practices for the genetic improvement of Malaysian indica rice. Full article

In the last three decades, the concept of soil humic substances has been questioned in two main directions. Misinterpretations of CP MAS¹³C NMR spectroscopy led to the conclusion that soil organic matter is mainly aliphatic, questioning the theory of polymerization of humic substances from phenolic molecules. Conversely, some critics of humic substances assume that a great proportion of aromatic soil organic carbon originates from fire-affected carbon, often termed as black carbon (BC). However, the determination of BC in soil by two widely applied methods, the benzene polycarboxylic acid marker method and the UV method, is not reliable and seems to strongly overestimate the BC content of soils. The concept of humic substances continues to be relevant today. The polymerization of phenolic molecules that originate from the degradation of lignin or synthesis by microorganisms may lead to humic substances which can incorporate a variety of organic and inorganic molecules and elements. The incorporation, e.g., of triazines or surfactants into the humic matrix, leading to bound residues, illustrates that humic substances are important to explain central reactions in soil. Humic substances are also important to understand the availability of plant nutrients in soil, including P, Fe, and Cu, and they may have a direct effect on the growth of higher plants in soil. Therefore, there are good reasons to reformulate or to further develop the concepts and models of humic substances introduced and developed by M. Schnitzer, W. Flaig, W. Ziechmann, and F.J. Stevenson. Full article

Aquaponics is a recirculating technology that combines aquaculture with hydroponics. It allows nutrients from fish waste to feed plants and thus saves water and nutrients. However, there is a mismatch between the nutrients provided by the fish waste and plant needs. Because of this, some nutrients, notably N, tend to accumulate in the aquaponic water (APW or AP water). The aim of this study was to investigate how APW, which is depleted of P and K but still rich in N, could be further utilized. APW was used in a mesocosm and compared with APW from the same source that had been supplemented with macro-nutrients (complemented AP water or CAPW) and a hydroponic control (HC). Mizuna (M) and rocket salad (R) were used as short-cycle vegetable crops in a NFT system. The results revealed that the low production potential of APW was mainly caused by the lack of P and K. If these were supplemented, the yields were comparable to those in the HC. M yield in CAPW was significantly higher than that of HC, probably due to biostimulant effects connected to the organic components in the water as a result of fish farming. Water type, cultivation density, and intercropping significantly influenced the qualitative characteristics of the crop in terms of antioxidant compounds and minerals. Nitrate content in vegetables was lower than European regulation limits. The extended use of APW is viable if the missing nutrients are supplemented; this could be a strategy to increase the efficiency of water and nitrogen use, while further reducing environmental impact. Full article

In rainfed agricultural ecosystems in northwest China, improving soil fertility and reducing greenhouse gas (GHG) emissions are key factors for developing sustainable agriculture. This study determined the short-term effects of different biochar amendment rates on diurnal and seasonal variations of GHG emissions in the Loess Plateau to produce a background dataset that may be used to inform nutrient management guidelines for semiarid environments. Biochar produced by pyrolysis at 300–500 °C from maize straw was applied at rates of 0, 10, 20, 30, 40, and 50 t ha⁻¹ (T0, T1, T2, T3, T4, T5), respectively. The results indicated that in the first year after the application, T3, T4, and T5 treatments increased soil organic carbon (0–10 cm) by 54.7%, 56.3%, and 56.9% compared to the other treatments. In the first, year, biochar amendment decreased diurnal CH₄ and N₂O flux by an average of 17–119% compared to T0, among which T3 had the lowest mean value. T3 and T4 also had similar mean CO₂ flux, which was 33% lower than T0. Application of 30 t ha⁻¹ biochar produced the lowest cumulative CO₂ and N₂O emissions of 2300 and 4.07 kg h⁻¹, respectively. Biochar amendment showed no effect on grain yiel but reduced the global warming potential and GHG emission intensity by an average of 23% and 25%, respectively. The biochar application rate of 30 t ha⁻¹ under the conditions of this study may be an appropriate rate for improving soil C sequestration and mitigation of GHG emissions in the first year after its application to soils on semi–arid Loess Plateau. Full article

Novel methods to increase crop productivity are required to meet anticipated demands for food, feed, fiber, and fuel. It is becoming feasible to use modern sensors and data analysis techniques for predicting plant growth and productivity based on genomic, phenotypic, and environmental data. To design and construct crops that deliver desired traits requires trained personnel with scientific and engineering expertise as well as a variety of “soft” skills. To address these needs at Iowa State University, we developed a graduate specialization called “Predictive Plant Phenomics” (P3). Although some of our experiences may be unique, many of the specialization’s principles are likely to be broadly applicable to others interested in developing graduate training programs in plant phenomics. P3 involves transdisciplinary training and activities designed to develop communication, teambuilding, and management skills. To support students in this demanding and unique intellectual environment, we established a two-week boot camp before their first semester and founded a community of practice to support students throughout their graduate careers. Assessments show that P3 students understand the transdisciplinary training concepts, have formed a beneficial and supportive community, and interact with diverse faculty outside of their home departments. To learn more about the P3 program, visit www.predictivephenomicsinplants.iastate.edu. Full article

The sustainability of irrigated agriculture depends on the quality of irrigation water used. The electrolyte concentration (EC) of irrigation water may lead to the accumulation of salts in the root zone layers and affect the physiological functions of the crop by osmotic and ion toxicity effects. Further, the cationic and anionic composition of the water may alter the exchangeable cation composition of the soil as well as its pH. Because of the dominance of sodium salts in many sources of irrigation water, parameters related to sodium such as exchangeable sodium percentage (ESP) of soils and sodium adsorption ratio (SAR) of soil solutions have been commonly used to study the effects of sodium in irrigation water on soil structural stability. Quirk and Schofield’s concept of ‘threshold electrolyte concentration’ (TEC) has shown the importance of electrolytes in preventing the effects of sodium on soil structure. Based on this concept, several models have been proposed to relate ESP or SAR with EC to predict the possible impacts of irrigation water on soil structural stability. However, many research reports indicate that this relationship varies with soils, and a given model is not suitable for all types of soils. Further, the effects of potassium and magnesium in the processes leading to clay dispersion are disregarded in these models. This essay analyses all the factors involved in the structural failure of soils with different cationic composition, identifies the defects in these TEC models, and re-defines TEC on the basis of new insights on dispersive and flocculating charges of soils. This review does not deal with EC effects on crops nor the role of contaminant ions not involved with soil structural stability. Full article

Scanning technologies based on X-ray Computed Tomography (CT) have been widely used in many scientific fields including medicine, nanosciences and materials research. Considerable progress in recent years has been made in agronomic and plant science research thanks to X-ray CT technology. X-ray CT image-based phenotyping methods enable high-throughput and non-destructive measuring and inference of root systems, which makes downstream studies of complex mechanisms of plants during growth feasible. An impressive amount of plant CT scanning data has been collected, but how to analyze these data efficiently and accurately remains a challenge. We review statistical and computational approaches that have been or may be effective for the analysis of 3D CT images of plant roots. We describe and comment on different approaches to aspects of the analysis of plant roots based on images, namely, (1) root segmentation, i.e., the isolation of root from non-root matter; (2) root-system reconstruction; and (3) extraction of higher-level phenotypes. As many of these approaches are novel and have yet to be applied to this context, we limit ourselves to brief descriptions of the methodologies. With the rapid development and growing use of X-ray CT scanning technologies to generate large volumes of data relevant to root structure, it is timely to review existing and potential quantitative and computational approaches to the analysis of such data. Summaries of several computational tools are included in the Appendix. Full article

Ravenna grass (Tripidium ravennae) is a tall robust bunchgrass with potential as an energy crop. The aim was to investigate the variation of agronomic traits of Ravenna grass. Univariate analyses of traits were conducted on 95 plants from 2013 to 2017. The traits were: biomass yield per plant; C, N, and ash concentrations; leaf and culm sap sucrose concentrations; percentage seed set, and the number of caryopses per panicle. In 2013, the biomass yield averaged 0.21 ± 0.09 kg per plant (mean ± the standard deviation). In 2014 to 2017, the yield averaged from 3.9 ± 0.8 kg per plant to 7.5 ± 1.8 kg per plant. Carbon concentration was generally higher than other energy crops, while N and ash concentrations were generally lower. Leaf sap sucrose ranged from 24.4 ± 4.6 g kg⁻¹ in 2016 to 41.6 ± 7.6 g kg⁻¹ in 2013. Culm sap sucrose varied from approximately 1.6 to 2.1 times that of leaf sap depending upon the harvest year. The percentage seed set varied between years ranging from 37.2 ± 12.4% to 56.6 ± 9.8%, and the mean number of caryopses per panicle varied from 4,770 ± 2,000 to 11,470 ± 3,075. Full article

Resources are limited, thus improving resource use efficiency is a key objective for cereal-based cropping systems. This field study was carried out to quantify resource use efficiencies in selected C₃ and C₄ cereals under split nitrogen (N) application regimes. The study included the following treatments: six cereals (three C₃: wheat, oat, and barley; and three C₄: maize, millet, and sorghum) and four split N application regimes (NS₁ = full amount of N at sowing; NS₂ = half N at sowing + half N at first irrigation; NS₃ = ⅓ N at sowing + ⅓ N at first irrigation + ⅓ N at second irrigation; NS₄ = ¼ N at sowing + ¼ N at first irrigation + ¼ N at second irrigation + ¼ N at third irrigation). Results revealed that C₄ cereals out-yielded C₃ cereals in terms of biomass production, grain yield, and resource use efficiencies (i.e., radiation use efficiency (RUE) and nitrogen use efficiency (NUE)), while splitting N into three applications proved to be a better strategy for all of the selected winter and summer cereals. The results suggest that C₄ cereals should be added into existing cereal-based cropping systems and N application done in three installments to boost productivity and higher resource use efficiency to ensure food security for the burgeoning population. Full article

Global temperature increases will affect Fusarium head blight (FHB) levels in wheat (Triticum aestivum L.). A pressing question is whether current sources of resistance will be effective in a warmer environment. We evaluated phenotypic response to disease in 238 soft winter wheat breeding lines and cultivars grown in 2015–2016 and 2016–2017 under control and warmed (+3 °C) conditions. Warming was achieved with heating cables buried 3 cm in the rhizosphere. We measured heading date, plant height, yield, FHB rating, Fusarium damaged kernels (FDK), deoxynivalenol (DON), leaf blotch rating, powdery mildew rating and leaf rust rating. There were significant (p < 0.01) differences among genotypes for all traits measured. Genome-wide association study (GWAS) identified 19 and 10 significant SNPs in the control and warmed treatments, respectively. FDK and DON levels were often significantly (p < 0.05) higher in warmed than in control when we contrasted alleles at important quantitative trait locus (QTL) such as Fhb1, Rht-B1 and D1 and all vernalization and photoperiod loci. Increased rhizosphere temperature resulted in a significantly (p < 0.01) earlier heading date (~3.5 days) both years of the study. Rank correlation between warmed and control treatments was moderate (r = 0.56). Though encouraging, it indicates that selection for performance under warming should be carried out in a warmed environment. Full article

The disposal of sugar mill effluent is a serious matter of concern for the sugar industry. In this regard, the dilution of sugar mill effluent in combination with plant growth promoting rhizobacteria (PGPR) might be a viable option for improving crop growth. In this study, we evaluated the potential of diluted sugar mill effluent (SME) and PGPR to improve maize (Zea mays L.) performance. Seeds of a maize hybrid (Pioneer 1543) were sown in 20 kg soil-filled pots. The pots were irrigated with various sugar mill effluent concentrations (viz. 0, 15%, 30%, 45%, 60%, 75% and 100% v/v). The results indicated that application of SME up to a concentration of 75% improved the stay-green, leaf emergence, growth and productivity of maize. However, the application of SME at a concentration of 100% was detrimental for maize plants and decreased the maize growth. The application of PGPR was also beneficial for improvement in stay-green, leaf emergence, growth and productivity of maize as compared with control (no PGPR application). In conclusion, the use of SME at concentration of 75% in combination with PGPR was the most effective method for improvement in stay-green, leaf emergence, growth and productivity of maize. Full article

To assess the influence of drought stress on the growth and nitrogen nutrition status of pakchoi (Brassica campestris ssp. Chinensis L.) at different nitrogen (N) levels, the changes in N accumulation and enzyme activities involved in N assimilation were investigated. The drought was induced by adding polyethylene glycol (PEG) under hydroponic culture conditions. Pakchoi seedlings were exposed to a modified nutrient solution with different nitrogen concentration (N1, N2, and N3 represent 2, 9 and 18 mM NaNO₃, respectively) and osmotic potential (W1, W2 and W3 represent 0, 60 and 120 g·L⁻¹ PEG 6000) in a full factorial, replicated randomized block design. A short time (seven days) of drought stress caused a significant decline in plant water content, transpiration rate, shoot biomass and shoot nitrogen concentration. Increasing N availability considerably alleviate drought stress by increasing the content of total free amino acids in the roots, promoting the acceleration of root biomass accumulation, and improving the activities of nitrate reductase (NR; EC 1.7.1.1) and glutamine synthetase (GS; EC 6.3.1.2) which would reduce moisture limitations. The results suggested that pakchoi supplied with relative higher N had better growth performance under drought stress. Full article

Soil microbial abundance and diversity change constantly in continuous cropping systems, resulting in the prevalence of soil-borne pathogens and a decline in crop yield in solar greenhouses. To investigate the effects of rice straw and biochar on soil microbial abundance and diversity in soils with a history of continuous planting, three treatments were examined: mixed rice straw and biochar addition (RC), rice straw addition (R), and biochar addition (C). The amount of C added in each treatment group was 3.78 g kg⁻¹ soil. Soil without rice straw and biochar addition was treated as a control (CK). Results showed that RC treatment significantly increased soil pH, available nitrogen (AN), available phosphorus (AP), and potassium (AK) by 40.3%, 157.2%, and 24.2%, respectively, as compared to the CK soil. The amount of soil labile organic carbon (LOC), including readily oxidizable organic carbon (ROC), dissolved organic carbon (DOC), and light fraction organic carbon (LFOC), was significantly greater in the RC, R, and C treatment groups as compared to CK soil. LOC levels with RC treatment were higher than with the other treatments. Both rice straw and biochar addition significantly increased bacterial and total microbial abundance, whereas rice straw but not biochar addition improved soil microbial carbon metabolism and diversity. Thus, the significant effects of rice straw and biochar on soil microbial carbon metabolism and diversity were attributed to the quantity of DOC in the treatments. Therefore, our results indicated that soil microbial diversity is directly associated with DOC. Based on the results of this study, mixed rice straw and biochar addition, rather than their application individually, might be key to restoring degraded soil. Full article

Salinity is one of the major factors limiting avocado yield, primarily due to the high concentration of ions in irrigation water. An experiment was conducted on 2 year old avocado plants (Persea americana Mill.) cv. Hass, grafted onto Duke 7 clonal rootstock growing in pots, to determine the effect of salt stress on growth, as well as physiological and biochemical responses, and the effect of seaweed extract (Ascophyllum nodosum) on salinity stress. Treatments consisted of different types of irrigation water: distilled water, 9 mM NaCl water, distilled water + 2.25 mL of seaweed extract, 9 mM NaCl water + 2.25 mL of seaweed extract and, 9 mM NaCl water + 1.5 mL of seaweed extract. The irrigation treatment was applied every 15 days for 8 months. Treatments with salt reduced plant growth by approximately 50% of the fresh weight of all avocado plant tissues. Seaweed extract reduced the effects of abiotic stress only at an early stage, and increased potassium (K) and calcium (Ca) concentrations in leaves. Full article

Image-based evaluation of phenotypic traits has been applied for plant architecture, seed, canopy growth/vigor, and root characterization. However, such applications using computer vision have not been exploited for the purpose of assessing the coleoptile length and herbicide injury in seeds. In this study, high-throughput phenotyping using digital image analysis was applied to evaluate seed/seedling traits. Images of seeds or seedlings were acquired using a commercial digital camera and analyzed using custom-developed image processing algorithms. Results from two case studies demonstrated that it was possible to use image-based high-throughput phenotyping to assess seeds/seedlings. In the seedling evaluation study, using a color-based detection method, image-based and manual coleoptile length were positively and significantly correlated (p < 0.0001) with reasonable accuracy (r = 0.69–0.91). As well, while using a width-and-color-based detection method, the correlation coefficient was also significant (p < 0.0001, r = 0.89). The improvement of the germination protocol designed for imaging will increase the throughput and accuracy of coleoptile detection using image processing methods. In the herbicide study, using image-based features, differences between injured and uninjured seedlings can be detected. In the presence of the treatment differences, such a technique can be applied for non-biased symptom rating. Full article

Nitrogen (N) is one of the most important nutrients required for high productivity of the maize plant. In most farmers’ fields in Sub-Saharan Africa (SSA), there is low availability of N in the soil mainly due to continuous cultivation of the land, crop residues removal, little or no application of fertilizers and rapid leaching. There is a need to develop low N tolerant and adapted maize genotypes. Evaluation of maize genotypes under different nitrogen conditions would therefore be useful in identifying genotypes that combine stability with high yield potential for both stress and non-stress environment. Eighty maize hybrids were evaluated at Mbalmayo and Nkolbisson in Cameroon, during 2012 and 2013 minor and major cropping seasons across 11 environments under low and high N conditions. The objectives of the study were: (i) to determine the effect of genotype x environment interaction (G × E) on grain yield and yield stability of single cross maize hybrids across low N and optimum N environments and (ii) to identify genotypes to recommend for further use in the breeding program. Yield data of 80 hybrids were analyzed initially and the analysis of 20 best performing genotypes was further performed for a better visualization and interpretation of the results. Combined analysis of variance showed highly significant G × E effects for grain yield. The GGE biplot analysis divided the study area into three mega environments: one related to the major cropping season while the two others were related to the minor cropping season. The grain yield of the 20 highest yielding hybrids ranged from 4484.7 to 5198.3 kg ha⁻¹. Hybrid 1368 × 87036 was the highest yielding in the minor season while the most outstanding hybrid, TL-11-A-1642-5 × 87036 was the best for the major season. The latter hybrid showed the potential for production across environments and should therefore be further tested in multiple environments to confirm consistency of its high yield performance and stability, and to facilitate its release as a commercial hybrid. High yielding but not stable hybrids across environments could be recommended for the specific environments where they performed well. Full article

Sustainable development of the bioenergy industry will depend upon the amount and quality of bioenergy feedstock produced. Switchgrass (Panicum virgatum L.) is a model lignocellulosic bioenergy crop but critical information is lacking for improved management, growth, and development simulation model calibration. A field study was conducted near Ames, IA during 2012–2013 with the objective to evaluate upland (“Cave-in-Rock”, ‘Trailblazer’ and ‘Blackwell’) and lowland (“Kanlow” and “Alamo”) switchgrass ecotypes for harvest timing on morphology (i.e., phenology, leaf area index (LAI), and biomass yield). The experiment used a randomized complete block design, with three upland and two lowland varieties harvested at six dates annually. In both years, delaying harvest to later maturity increased biomass yield; lowland cultivars produced greater biomass yield (6.15 tons ha⁻¹) than upland ecotypes (5.10 tons ha⁻¹). Lowland ecotypes had delayed reproductive development compared with upland ecotypes. At the end of both growing seasons, upland ecotypes had greater mean stage count (MSC) than lowland ecotypes. “Cave-in-Rock” had greatest MSC and LAI, but did not produce the greatest biomass. Relationships were nonlinear between MSC and biomass yield, with significant cultivar–year interaction. The relationship between biomass yield and MSC will be useful for improving switchgrass, including cultivar selection, fertilizer application, and optimum harvest time. Full article

Many late-season physiological traits affect grain yield in wheat, either directly or indirectly. However, information on the genetic control of yield-related traits is still limited. In this study, we aimed to identify quantitative trait loci (QTL) for canopy temperature and chlorophyll content index during anthesis (CTa and CCIa, respectively), the mid grain-filling stage (CTg1 and CCIg1, respectively), and the late grain-filling stage (CTg2 and CCIg2, respectively) as well as for plant height (PH), thousand kernels weight (TKW), and grain yield (GY) using genome-wide linkage mapping. To this end, a double haploid population derived from a cross between two high yielding wheat cultivars, UI Platinum and SY Capstone, was phenotyped in four irrigated environments and genotyped using the wheat 90K iSelect platform and simple sequence repeats. The genotypic data were used to construct a high-density genetic map of 43 linkage groups (LGs) with a total length of 3594.0 cm and a marker density of 0.37 cm. A total of 116 QTL for all nine traits was detected on 33 LGs, spreading to all wheat chromosomes, except for Chr. 7D. Of these, six QTL (CTa.ui-4B.1, Q.CTg1.ui-5B-2.1, Q.CTg2.ui-6B.1, Q.PH.ui-6A-2.1, Q.TKW.ui-2D-1, and Q.GY.ui-6B) were consistently detected in more than three irrigated environments, called as stable QTL. Additionally, we identified 26 QTL clusters for more than two traits, of which the top four were located on Chromosomes 4A-1, 1B-1, 5B-2, and 2D-1. Overall, the stable QTL significantly related with grain yield, QTL clusters, and linked molecular markers identified in this study, may be useful in marker-assisted selection in early generation and early growth stage for grain yield improvement. Full article

Among one of the many challenges in implementing precision irrigation is to obtain an accurate characterization of the soil water content (SWC) across spatially variable fields along the crop growing season. The accuracy of characterizing SWC has been tested primarily on a small-scale and has received little attention from the scientific community at the field scale. Hence, the objective of this study was to assess the characterization of the spatial distribution of soil water content at the field scale by the apparent electrical conductivity (ECa). In evaluating the current aim, ECa survey was compared against repeated measurements of SWC at five depths using neutron probe. Results showed that mean SWC was different across ECa derived management zones, which indicates that on a macro-scale, soil ECa could effectively characterize the mean differences in SWC across management zones. Results also showed that deep ECa (0–150 cm) survey outperformed shallow survey (0–75 cm). Considering other soil properties, such as organic matter content and salt content, further improved the relationship between SWC and ECa. Full article

Interspecific hybridization has been attempted to combine the heat and drought of Poa arachnifera Torr. with the turf quality characteristics of several Poa species. Confirmation of an F₁ hybrid through morphological analysis of vegetative and flowering characteristics is often time consuming and ambiguous. Ent-kaurene oxidase (KO) has been sequenced in rice, barley, and wheat. In rice, each of the five copies of KO gene has unique lengths for the first intron. Conserved intron spanning primers (CISP) can be used as a DNA marker to exploit variations of intron lengths that flank conserved gene sequences. In the present study, we developed CISP to sequence partial genomic fragments of the KO gene from seven Poa species. Through sequence analysis, species-specific primers were also developed to produce co-dominant markers that can be used to identify interspecific hybrids between Texas bluegrass and six other Poa species used in the present study. Full article