Other Plant Sciences Commons^™

Dt2 Is A Gain-Of-Function Mads-Domain Factor Gene That Specifies Semideterminacy In Soybean, Jieqing Ping, Yunfeng Liu, Lianjun Sun, Meixia Zhao, Yinghui Li, Zongxiang Tang, Hanh Nguyen, Zhixi Tian, Lijuan Qiu, Randall L. Nelson, Thomas E. Clemente, James Specht, Jianxin Ma

Department of Agronomy and Horticulture: Faculty Publications

Similar to Arabidopsis thaliana, the wild soybeans (Glycine soja) and many cultivars exhibit indeterminate stem growth specified by the shoot identity gene Dt1, the functional counterpart of Arabidopsis TERMINAL FLOWER1 (TFL1). Mutations in TFL1 and Dt1 both result in the shoot apical meristem (SAM) switching from vegetative to reproductive state to initiate terminal flowering and thus produce determinate stems. A second soybean gene (Dt2) regulating stem growth was identified, which, in the presence of Dt1, produces semideterminate plants with terminal racemes similar to those observed in determinate plants. Here, we report positional …

Eqtl Networks Reveal Complex Genetic Architecture In The Immature Soybean Seed, Yung-Tsi Bolon, D. Hyten, James H. Orf, Carroll P. Vance, Gary J. Muehlbauer

Department of Agronomy and Horticulture: Faculty Publications

The complex network of regulatory factors and interactions involved in transcriptional regulation within the seed is not well understood. To evaluate gene expression regulation in the immature seed, we utilized a genetical genomics approach on a soybean [Glycine max (L.) Merr.] recombinant inbred line (RIL) population and produced a genome-wide expression quantitative trait loci (eQTL) dataset. The validity of the dataset was confirmed by mapping the eQTL hotspot for flavonoid biosynthesis-related genes to a region containing repeats of chalcone synthase (CHS) genes known to correspond to the soybean inhibitor locus that regulates seed color. We then identified eQTL for …

Nebraska’S Groundwater Legacy: Nitrate Contamination Beneath Irrigated Cropland, Mary E. Exner Spaulding, Aaron J. Hirsh, Roy F. Spalding

Department of Agronomy and Horticulture: Faculty Publications

A 31 year record of ~44,000 nitrate analyses in ~11,500 irrigation wells was utilized to depict the decadal expansion of groundwater nitrate contamination (N>10 mg/L) in the irrigated corn-growing areas of eastern and central Nebraska and analyze long-term nitrate concentration trends in 17 management areas (MAs) subject to N fertilizer and budgeting requirements. The 1.3 M contaminated hectares were characterized by irrigation method, soil drainage, and vadose zone thickness and lithology. The areal extent and growth of contaminated groundwater in two predominately sprinkler-irrigated areas was only ~20% smaller beneath well-drained silt loams with thick clayey-silt unsaturated layers and unsaturated …

Multilocation Corn Stover Harvest Effects On Crop Yields And Nutrient Removal, Douglas L. Karlen, Stuart J. Birrell, Jane M.F. Johnson, Shannon L. Osborne, Thomas E. Schumacher, Gary E. Varvel, Richard B. Ferguson, Jeff M. Novak, James R. Frederick, John M. Baker, John A. Lamb, Paul R. Alder, Greg W. Roth, Emerson D. Nafziger

Department of Agronomy and Horticulture: Faculty Publications

Corn (Zea mays L.) stover was identified as an important feedstock for cellulosic bioenergy production because of the extensive area upon which the crop is already grown. This report summarizes 239 site-years of field research examining effects of zero, moderate, and high stover removal rates at 36 sites in seven different states. Grain and stover yields from all sites as well as N, P, and K removal from 28 sites are summarized for nine longitude and six latitude bands, two tillage practices (conventional vs no tillage), two stoverharvest methods (machine vs calculated), and two crop rotations {continuous corn (maize) …

Do Cover Crops Increase Or Decrease Nitrous Oxide Emissions? A Meta-Analysis, Andrea D. Basche, F. E. Miguez, T. C. Kaspar, M. J. Castellano

Department of Agronomy and Horticulture: Faculty Publications

There are many environmental benefits to incorporating cover crops into crop rotations, such as their potential to decrease soil erosion, reduce nitrate (NO₃) leaching, and increase soil organic matter. Some of these benefits impact other agroecosystem processes, such as greenhouse gas emissions. In particular, there is not a consensus in the literature regarding the effect of cover crops on nitrous oxide (N₂O) emissions. Compared to site-specific studies, meta-analysis can provide a more general investigation into these effects. Twenty-six peer-reviewed articles including 106 observations of cover crop effects on N₂O emissions from the soil surface …

Challenges And Opportunities In Transdisciplinary Science: The Experience Of Next Generation Scientists In An Agriculture And Climate Research Collaboration, Andrea D. Basche, Gabrielle E. Roesch-Mcnally, Lindsay A. Pease, Christopher D. Eidson, Guy Bou Lahdou, Mike W. Dunbar, Trevor J. Frank, Laura Frescoln, Lei Gu, Ryan Nagelkirk, Jose Pantoja, Adam K. Wilke

Department of Agronomy and Horticulture: Faculty Publications

Agriculture in the twenty-first century faces unprecedented challenges from increasing climate variability to growing demands on natural resources to globalizing economic markets. These emerging agricultural issues, spanning both human and natural dimensions, are uniquely formulated, exceedingly complex, and difficult to address within existing disciplinary domains (Eigenbrode et al. 2007; Reganold et al. 2011; Foley et al. 2005; Hansen et al. 2013). Therefore, the next generation of scientists working on these issues must not only be highly trained within a disciplinary context but must also have the capacity to collaborate with others to solve systems-level problems.

The Carbon-Nitrogen Balance Of The Nodule And Its Regulation Under Elevated Carbon Dioxide Concentration, Marc Libault

Department of Agronomy and Horticulture: Faculty Publications

Legumes have developed a unique way to interact with bacteria: in addition to preventing infection from pathogenic bacteria like any other plant, legumes also developed a mutualistic symbiotic relationship with one gender of soil bacteria: rhizobium. This interaction leads to the development of a new root organ, the nodule, where the differentiated bacteria fix for the plant the atmospheric dinitrogen (atmN₂). In exchange, the symbiont will benefit from a permanent source of carbon compounds, products of the photosynthesis. The substantial amounts of fixed carbon dioxide dedicated to the symbiont imposed to the plant a tight regulation of the …

Corn (Zea Mays) Emergence And Early Growth As Influenced By Tansymustard (Descurainia Pinnata) Residue, Rodrigo Werle, Leonardo L. Perez, Lowell D. Sandell, John L. Lindquist

Department of Agronomy and Horticulture: Faculty Publications

Farmers have observed reduced early growth of corn in the presence of tansymustard [Descurainia pinnata (Walt.) Britt.], a winter annual weed of the Brassicaceae family. A greenhouse study was conducted to evaluate the effect of tansymustard on corn (Zea mays L.) emergence and early growth. Treatments included corn growing in pots with only tansymustard root residue, with only tansymustard shoot residue, and with whole tansymustard plant residue (roots plus shoots); tansymustard plants treated with glyphosate 2 days after planting (DAP) corn seeds; and a control (no tansymustard residue). Corn emergence was recorded and biomass measured after 4 weeks of …

Evaluation Of Soilless Media Sensors For Managing Winter-Time Greenhouse Strawberry Production Using A Capmat System, George E. Meyer, Ellen T. Paparozzi, Stacy A. Adams, Diego Scacalossi Voltan

Department of Agronomy and Horticulture: Faculty Publications

It is important for a greenhouse strawberry grower to know that their capillary mat (CapMat™) fertigation system is working correctly and that plants are receiving the correct amounts of water and fertilizer. Pots with soilless mix are not expected to hold more than 70% water on a volumetric basis. Pots with less than 40% water content continuously are not supplied enough water and nutrients to the plants. Typically, pots located near the manifold distribution system get a little more water than those at the other locations, but water use will really vary according to the factors listed above as well …

Remote Estimation Of Nitrogen And Chlorophyll Contents In Maize At Leaf And Canopy Levels, Michael Schlemmer, Anatoly A. Gitelson, James S. Schepers, Richard B. Ferguson, Y. Peng, J. Shanahan, Donald Rundquist

Department of Agronomy and Horticulture: Faculty Publications

Leaf and canopy nitrogen (N) status relates strongly to leaf and canopy chlorophyll (Chl) content. Remote sensing is a tool that has the potential to assess N content at leaf, plant, field, regional and global scales. In this study, remote sensing techniques were applied to estimate N and Chl contents of irrigated maize (Zea mays L.) fertilized at five N rates. Leaf N and Chl contents were determined using the red-edge chlorophyll index with R2 of 0.74 and 0.94, respectively. Results showed that at the canopy level, Chl and N contents can be accurately retrieved using green and red-edge Chl …

Optimal Copper Supply Is Required For Normal Plant Iron Deficiency Responses, Brian M. Waters, Laura C. Armbrust

Department of Agronomy and Horticulture: Faculty Publications

Iron (Fe) and copper (Cu) homeostasis are tightly linked across biology. Understanding crosstalk between Fe and Cu nutrition could lead to strategies for improved growth on soils with low or excess metals, with implications for agriculture and phytoremediation. Here, we show that Cu and Fe nutrition interact to increase or decrease Fe and/or Cu accumulation in leaves and Fe uptake processes. Leaf Cu concentration increased under low Fe supply, while high Cu lowered leaf Fe concentration. Ferric reductase activity, an indicator of Fe demand, was inhibited at insufficient or high Cu supply. Surprisingly, plants grown without Fe were more susceptible …

A Study Of The Historical Development And Horticultural Practices Of A Public Garden, Steven J. Nosal

Department of Agronomy and Horticulture: Dissertations, Theses, and Student Research

This project examined the historical development and the horticultural practices in the public gardens of the Parks Department in Lincoln, Neb. It concentrates on one particular place to illustrate development and practice issues: the Sunken Gardens. This garden was built during the Depression in the 1930s as a place of beauty for all of the citizens of Lincoln. Although the Sunken Garden covers only 1.5 acres, it has remained a very important part of Nebraska's capital city for more than 64 years.

Automated Conserved Non-Coding Sequence (Cns) Discovery Reveals Differences In Gene Content And Promoter Evolution Among Grasses, Gina Marie Turco, James C. Schnable, Brent S. Pedersen, Michael Freeling

Department of Agronomy and Horticulture: Faculty Publications

Conserved non-coding sequences (CNS) are islands of non-coding sequence that, like protein coding exons, show less divergence in sequence between related species than functionless DNA. Several CNSs have been demonstrated experimentally to function as cis-regulatory regions. However, the specific functions of most CNSs remain unknown. Previous searches for CNS in plants have either anchored on exons and only identified nearby sequences or required years of painstaking manual annotation. Here we present an open source tool that can accurately identify CNSs between any two related species with sequenced genomes, including both those immediately adjacent to exons and distal sequences separated by …

Rate Of Shattercane × Sorghum Hybridization In Situ, Jared J. Schmidt, Jeffrey F. Pedersen, Mark L. Bernards, John L. Lindquist

Department of Agronomy and Horticulture: Faculty Publications

Cultivated sorghum [Sorghum bicolor (L.) Moench subsp. bicolor] can interbreed with a feral weedy relative shattercane [S. bicolor nothosubsp. drummondii (Steud.) de Wet ex Davidse]. Traits introduced from cultivated sorghum could contribute to the invasiveness of a shattercane population. An experiment was conducted to determine the potential for pollenmediated gene flow from grain sorghum to shattercane. Shattercane with juicy midrib (dd) was planted in soybean [Glycine max (L.) Merr.] fields during 2 yr in concentric arcs at varying distances from a 0.39 ha sorghum pollen source with dry midrib (DD). The arcs …

A Floristic Analysis And Comparison Of Plant Communities In Harlan County, Nebraska, Naomi D. Hastings, Steven J. Rothenberger

Transactions of the Nebraska Academy of Sciences and Affiliated Societies

Historically, the Republican River Valley in Harlan County, Nebraska, has been extensively explored, but the county flora has continually been under-represented and has been found to lack records for plants that are considered common. During the 2009 growing season, a taxonomic study and comparison of 3 different sites in Harlan County was made. Site #1 was pastureland and Sites #2 and #3 were both riparian, located on the north side of the Republican River. Collections were pressed, dried, identified, and deposited in the University of Nebraska at Kearney Herbarium (NEBK). Forty-eight new county records were verified, in­cluding cutleaf-cucumber (Cyclanthera …

Evaluating The Success Of Seed Sowing In A New England Grassland, Chad C. Jones, Glenn D. Dreyer, Nels Barrett

Botany Faculty Publications

Grassland habitat is declining in the northeastern United States, leading to a decline in associated native species. Consequently, there is considerable interest by land managers in conserving and restoring grassland habitats in the Northeast. However, unlike the Great Plains and Europe, quantitative monitoring of restoration sites is uncommon, making it difficult to improve new restoration projects. Here we evaluate a grassland restoration in Waterford, Connecticut to determine if mechanical clearing of woody vegetation combined with sowing 23 native grasses and forbs led to successful establishment of these species. We also compared cover, diversity, and colonization by exotic and woody species …

Arbuscular Mycorrhizal Fungi Differ In Their Ability To Regulate The Expression Of Phosphate Transporters In Maize (Zea Mays L.), Hui Tian, Rhae A. Drijber, Xiaolin Li, Daniel N. Miller, Brian J. Wienhold

Department of Agronomy and Horticulture: Faculty Publications

Previous studies have found that some phosphate (Pi) starvation inducible transporter genes are downregulated and arbuscular mycorrhizal (AM) inducible Pi transporter genes are upregulated in maize roots associated with the fungus Glomus intraradices. However, little is known about the functional diversity of different AM fungal species in influencing the expression of Pi transporters in maize roots. Here, we studied the expression of two Pi transporter genes ZEAma:Pht1;3 (Pi starvation inducible) and ZEAma:Pht1;6 (AM inducible) in maize root colonized by different AM fungal inoculants. Non-mycorrhizal maize, maize colonized by Glomus deserticola (CA113), Glomus intraradices (IA506), Glomus mosseae (CA201), Gigaspora gigantea …

Differential Allocation To Photosynthetic And Non-Photosynthetic Nitrogen Fractions Among Native And Invasive Species, Jennifer L. Funk, Lori A. Glenwinkle, Lawren Sack

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Invasive species are expected to cluster on the “high-return” end of the leaf economic spectrum, displaying leaf traits consistent with higher carbon assimilation relative to native species. Intra-leaf nitrogen (N) allocation should support these physiological differences; however, N biochemistry has not been examined in more than a few invasive species. We measured 34 leaf traits including seven leaf N pools for five native and five invasive species from Hawaii under low irradiance to mimic the forest understory environment. We found several trait differences between native and invasive species. In particular, invasive species showed preferential N allocation to metabolism (amino acids) …

Sub1a-Mediated Submergence Tolerance Response In Rice Involves Differential Regulation Of The Brassinosteroid Pathway, Aaron J. Schmitz, Jing J. Folsom, Yusuke Jikamaru, Pamela C. Ronald, Harkamal Walia

Department of Agronomy and Horticulture: Faculty Publications

Submergence 1A (SUB1A), is an ethylene response factor (ERF) that confers submergence tolerance in rice (Oryza sativa) via limiting shoot elongation during the inundation period. SUB1A has been proposed to restrict shoot growth by modulating gibberellic acid (GA) signaling.

Our transcriptome analysis indicated that SUB1A differentially regulates genes associated with brassinosteroid (BR) synthesis during submergence. Consistent with the gene expression data, the SUB1A genotype had higher brassinosteroid levels after submergence compared to the intolerant genotype. Tolerance to submergence can be activated in the intolerant genotype by pretreatment with exogenous brassinolide, which results in restricted shoot elongation …

Erratum To: Identification And Characterization Of Four Missense Mutations In Brown Midrib12 (Bmr12), The Caffeic Acid O-Methyltranferase (Comt) Of Sorghum, Scott E. Sattler, Nathan A. Palmer, Ana Saballos, Ann M. Greene, Zhanguo Xin, Gautam Sarath, Wilfred Vermerris, Jeffrey F. Pedersen

Department of Agronomy and Horticulture: Faculty Publications

The original version of this article unfortunately contained some mistakes. The name of the enzyme “Caffeic O-methyltransferase” should be read as “Caffeic acid O-Methyltranferase” throughout the paper, including the title.

On Table 2, the fifth column should have been captured as part of the sub-heading “Unpretreated”. The corrected version is shown on the next page.

Table 2 Variation in Klason lignin content and glucose yields obtained after enzymatic saccharification at 50 °C at 60 FPU/ g cellulase of native (unpretreated) stover after 4, 20, and 96 h, and of pretreated stover after 24 h

Mapping Soybean Aphid Resistance Genes In Pi 567598b, Carmille Bales, Guorong Zhang, Menghan Liu, Clarice Mensah, Cuihua Gu, Qijian Song, D. L. Hyten, P. B. Cregan, Dechun Wang

Department of Agronomy and Horticulture: Faculty Publications

The soybean aphid (Aphis glycines Matsumura) has been a major pest of soybean [Glycine max (L.) Merr.] in North America since it was first reported in 2000. Our previous study revealed that the strong aphid resistance of plant introduction (PI) 567598B was controlled by two recessive genes. The objective of this study was to locate these two genes on the soybean genetic linkage map using molecular markers. A mapping population of 282 F_4:5 lines derived from IA2070 X E06902 was evaluated for aphid resistance in a field trial in 2009 and a greenhouse trial in 2010. Two …

Unleashing The Potential Of The Root Hair Cell As A Single Plant Cell Type Model In Root Systems Biology, Zhenzhen Qiao, Marc Libault

Department of Agronomy and Horticulture: Faculty Publications

Plant root is an organ composed of multiple cell types with different functions.This multicellular complexity limits our understanding of root biology because -omics studies performed at the level of the entire root reflect the average responses of all cells composing the organ. To overcome this difficulty and allow a more comprehensive understanding of root cell biology, an approach is needed that would focus on one single cell type in the plant root. Because of its biological functions (i.e., uptake of water and various nutrients; primary site of infection by nitrogen-fixing bacteria in legumes), the root hair cell is an attractive …

Leaf Traits Within Communities: Context May Affect The Mapping Of Traits To Function, Jennifer L. Funk, William K. Cornwell

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

The leaf economics spectrum (LES) has revolutionized the way many ecologists think about quantifying plant ecological trade-offs. In particular, the LES has connected a clear functional trade-off (long-lived leaves with slow carbon capture vs. short-lived leaves with fast carbon capture) to a handful of easily measured leaf traits. Building on this work, community ecologists are now able to quickly assess species carbon-capture strategies, which may have implications for community-level patterns such as competition or succession. However, there are a number of steps in this logic that require careful examination, and a potential danger arises when interpreting leaf-trait variation among species …

The Clubroot Pathogen (Plasmodiophora Brassicae) Influences Auxin Signaling To Regulate Auxin Homeostasis In Arabidopsis, Linda Jahn, Stefanie Mucha, Sabine Bergmann, Cornelia Horn, Paul E. Staswick, Bianka Steffens, Johannes Siemens, Jutta Ludwig-Müller

Department of Agronomy and Horticulture: Faculty Publications

The clubroot disease, caused by the obligate biotrophic protist Plasmodiophora brassicae, affects cruciferous crops worldwide. It is characterized by root swellings as symptoms, which are dependent on the alteration of auxin and cytokinin metabolism. Here, we describe that two different classes of auxin receptors, the TIR family and the auxin binding protein 1 (ABP1) in Arabidopsis thaliana are transcriptionally upregulated upon gall formation. Mutations in the TIR family resulted in more susceptible reactions to the root pathogen. As target genes for the different pathways we have investigated the transcriptional regulation of selected transcriptional repressors (Aux/IAA) and transcription …

The Physiology Of Invasive Plants In Low-Resource Environments, Jennifer L. Funk

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

While invasive plant species primarily occur in disturbed, high-resource environments, many species have invaded ecosystems characterized by low nutrient, water, and light availability. Species adapted to low-resource systems often display traits associated with resource conservation, such as slow growth, high tissue longevity, and resource-use efficiency. This contrasts with our general understanding of invasive species physiology derived primarily from studies in high-resource environments. These studies suggest that invasive species succeed through high resource acquisition. This review examines physiological and morphological traits of native and invasive species in low-resource environments. Existing data support the idea that species invading low-resource environments possess traits …

Factors Influencing The Establishment And Survival Of Native Hardwood Tree Seedlings Of The Kentucky Inner Bluegrass Blue Ash-Oak Savanna-Woodland, James D. Shaffer

Theses and Dissertations--Biology

Historically, the Kentucky Inner Bluegrass blue ash-oak savanna-woodland was the primary ecosystem of the Inner Bluegrass Region (IBR) of Kentucky. After European settlement, the majority (>99%) of Bluegrass savanna was converted to agricultural and urban land uses. Currently remnant savanna tree species are failing to recruit. Therefore, a long-term restoration ecology project researching competition and disturbance on seedling establishment, survival, and growth has been established at Griffith Woods (the largest remaining savanna in Kentucky) in Harrison Co., KY. Fourteen native hardwood tree species (a total of 6,168 seedlings) have been experimentally planted. Light, soil, surrounding vegetation, and herbivory, factors …

Immunodetection Of Triticum Mosaic Virus By Das- And Dac-Elisa Using Antibodies Produced Against Coat Protein Expressed In Escherichia Coli: Potential For High-Throughput Diagnostic Methods, Satyanarayana Tatineni, Gautam Sarath, Dallas Seifers, Roy C. French

Department of Agronomy and Horticulture: Faculty Publications

Triticum mosaic virus (TriMV), an economically important virus infecting wheat in the Great Plains region of the USA, is the type species of the Poacevirus genus in the family Potyviridae. Sensitive and high-throughput serology-based detection methods are crucial for the management of TriMV and germplasm screening in wheat breeding programs. In this study, TriMV coat protein (CP) was expressed in Escherichia coli, and polyclonal antibodies were generated against purified soluble native form recombinant CP (rCP) in rabbits. Specificity and sensitivity of resulting antibodies were tested in Western immuno-blot and enzyme-linked immunosorbent assays (ELISA). In direct antigen coating (DAC)-ELISA, …

Soil Sorptivity Enhancement With Crop Residue Accumulation In Semiarid Dryland No-Till Agroecosystems, T.M. Shaver, G. A. Peterson, Laj R. Ahuja, D.G. Westfall

Department of Agronomy and Horticulture: Faculty Publications

Water capture and precipitation use efficiency are of great importance in dryland cropping systems because the world's dependence on food produced in dryland areas continues to increase. Growing season evapotranspiration potential greatly exceeds growing season precipitation rates in dryland areas, creating a water deficit for crops. Management practices that positively impact soil physical properties increase the potential for soils to capture water. One way to assess the ability of soils to capture water is through the measurement of sorptivity. Sorptivity is defined as the cumulative infiltration proportionality constant and is governed by surface soil physical properties such as texture, degree …

Enzyme Activity In Wheat Breeding Lines Derived From Matings Of Low Polyphenol Oxidase Parents, Somrudee Nilthong, Robert A. Graybosch, P. Stephen Baenziger

Department of Agronomy and Horticulture: Faculty Publications

Polyphenol oxidase (PPO) in grain plays a major role in time-dependent discoloration of wheat (Triticum aestivum L.) products, especially fresh noodles. Breeding wheat cultivars with low or nil PPO activity can reduce undesirable product darkening. The low PPO line PI 117635 was crossed to two low PPO wheats, IDO580 and ‘IDO377s’, to determine whether matings between wheats with low levels of grain PPO would result in complementation, such that lines with still lower or nil PPO would be generated. Progeny in a population derived from PI 117635/ IDO580 displayed no variation in PPO activity. In the F_3:4 populations …

Evaluation Of Public Sweet Sorghum A-Lines For Use In Hybrid Production, Jeffrey F. Pedersen, Scott E. Sattler, William F. Anderson

Department of Agronomy and Horticulture: Faculty Publications

A fundamental need for commercialization of sweet sorghum [Sorghum bicolor (L.) Moench] as a bioenergy crop is an adequate seed supply, which will require development of hybrid varieties using dwarf seed-parent lines. A set of six public sweet sorghum A-lines (Dwarf Kansas Sourless, KS9, N36, N38, N39, and N4692) were crossed with a set of six public sweet sorghum cultivars (Brawley, Kansas Collier, Dale, Sugar Drip, Waconia, and Wray). Grain, fiber, and sugar yields were determined, and conversion formulas were applied to estimate ethanol yields. Hybrids were grown in fields at Ithaca, NE, USA, in 1983– 1984 fertilized with …