Influence of Air Temperature on the Stability of Cytoplasmic Male Sterility (CMS) in Maize (Zea mays L.) Heidrun Bückmann, Katja Thiele, Alexandra Hüsken, Joachim Schiemann Institute for Biosafety in Plant Biotechnology www.jki.bund.de Background Reduction of Cross-Pollination = Biological Confinement Cytoplasmic Male Sterility in Maize Mutation in the mitochondrial Genome (maternally inherited ) Nucleare restorer of fertility genes (Rf-Gene) Male Sterility (no or no vital pollen) Institute for Biosafety in Plant Biotechnology Fertile pollen www.jki.bund.de Background Cytoplasmic Male Sterility (CMS) Types of cytoplasm Cytoplasm T: Cytoplasm C: Cytoplasm S: - high level of stability - Rf1 and Rf2 required for complete restoration - Rf8 as replacement for Rf1, partly restoration -medium stability -Rf4 or Rf5 and/or Rf6 required for restoration - CMS low stability - Rf3 or Rf9 sufficient for restoration - many undefined allels of Rf-genes available Characteristics of CMS Partly restoration of fertility possible for all types of S-, C- and T-cytoplasm Clear rules of the restoration process still unknown environmental impact undefined quantity of Rf genes involved mutation of the nuclear genome: new Rf-genes Rf-genes can be environmental sensitive Institute for Biosafety in Plant Biotechnology www.jki.bund.de Field experiments Assessing the reliability of CMS maize as a biological confinement method in different environments in Germany Heidrun Bückmann, Alexandra Hüsken, Joachim Schiemann (2013): Applicability of cytoplasmic male sterility (CMS) as a reliable biological confinement method for the cultivation of genetically modified maize in Germany, Journal of Agricultural Science and Technology A 3, 385-403 Topics: • Stabilty of the CMS trait in Maize hybrids • Cross-pollination rate and distribution of CMS maize pollen • Extend of cross-pollination reduction compared to conventional varieties Experiments: • Big plots (3300 m²) • 3 locations • 2 years CMS maize hybrids: • DSP2 (T-cytoplasm) • Torres (S-cytoplasm) • Zidane (S-cytoplasm) Conventional hybrid: Delitop Pollen acceptor: White maize DSP 17007 Institute for Biosafety in Plant Biotechnology sterile tassel No pollen partly restored tassel fertile tassel Vital pollen? Vital pollen? www.jki.bund.de Tassel characteristics, Pollen developement and vitality CMShybride location Hötzum DSP2 Groß Lüsewitz Freising Hötzum Torres Groß Lüsewitz Freising Hötzum Zidane Groß Lüsewitz Freising tassels [%] sterile partly rest. fertile sterile partly rest. fertile sterile partly rest. fertile sterile partly rest. fertile sterile partly rest. fertile sterile partly rest. fertile sterile partly rest. fertile sterile partly rest. fertile sterile partly rest. fertile Institute for Biosafety in Plant Biotechnology pollen [%] 2009 2010 96 3 1 5 87 8 n.s. n.s. n.s. 1 99 0 0 80 20 n.s. n.s. n.s. 0 58 49 0 51 49 n.s. n.s. n.s. 98 1 1 76 22 2 96 4 0 7 92 1 1 98 1 3 97 0 0 59 41 0 87 13 0 98 2 no few many no few many no few many no few many no few many no few many No few many no few many no few many MKS [%] 2009 2010 96 3 1 10 78 12 n.s. n.s. n.s. 13 87 0 12 87 1 n.s. n.s. n.s. 0 33 67 0 57 43 n.s. n.s. n.s. 99 1 0 76 24 0 96 4 0 39 61 0 3 96 1 100 0 0 0 87 13 0 87 13 3 95 2 2009 2010 0,22 8,60 40 0,02 81 1,67 0,71 0,00 MKS = mean kernel set number of developed kernels per corn cob in relation to a complete fertilized corn cob at self-pollinated plants 2 0,03 3,09 1 0,23 0,43 20 1,63 44 6,89 44 4,11 3 www.jki.bund.de Cross-pollination [%] – 2009 Hötzum Groß Lüsewitz 48 m Delitop 48m Freising Delitop 48 m 36 36 36 24 24 24 12 12 12 0 3.5 10 20 30 40 50 48 m 60 70mm 70 DSP2 0 3.5 10 20 30 40 50 48 m 60 70mm 70 DSP2 0 3.5 10 36 36 24 24 24 12 12 12 20 30 40 50 48 m 60 70mm Torres 0 3.5 10 20 30 40 50 48 m 60 70mm 70 Torres 0 3.5 10 36 36 24 24 24 12 12 12 20 30 40 50 48 m 60 70mm Zidane 0 3.5 10 20 30 40 50 48 m 60 70mm 70 Zidane 0 3.5 10 36 36 24 24 24 12 12 12 20 30 40 50 60 70mm 70 0 3.5 10 20 30 Institute for Biosafety in Plant Biotechnology 40 50 60 70mm 70 30 40 50 0 3.5 10 60 70mm 70 DSP2 20 30 40 50 60 70mm 70 Torres 20 30 40 50 48 m 36 0 3.5 10 20 48 m 36 0 3.5 10 Cross-pollination rate [%] 48 m 36 0 3.5 10 Delitop 60 70mm 70 Zidane 20 30 40 50 60 70mm 70 www.jki.bund.de Monitoring in the Field 2010 Location: Hötzum, CMS hybrid: DSP2 Situation A: Situation B: partly restored and sterile plants at the same time in one plot growing delay of 14 days flowering at 2°C lower air temperatures all plants partly restored Climatical Influence or Seed Contamination? Test of seed contamination: all plants were CMS-T types A PCR Assay for Rapid Dircrimination of Sterile Cytoplasm Types in Maize Liu, Z., Peter, S. O., Long, M. Weingartner, U., Stamp, P. und Kaeser, O. ; Crop Sci. 42: 566-569 (2002) Institute for Biosafety in Plant Biotechnology www.jki.bund.de Air temperatures at flowering time Groß Lüsewitz Freising 25 25 20 20 20 15 3.5°C 10 5 temp. 2009 15 temp. 2010 10 5 0 Air Temperature [%] 25 Air Temperture [%] Air Temperature [°C] Hötzum mean temp (61-10) 0 June July August temp. 2009 temp. 2009 temp. 2010 temp. 2010 mean temp. (73-07) mean temp. (61-10) 15 10 5 0 June July August June July August regular main flowering later main flowering Institute for Biosafety in Plant Biotechnology www.jki.bund.de 1st Green House Experiment Influence of the Air Temperature on the Stability of Cytoplasmic Male Sterility (CMS) in Maize CMS maize hybrids • DSP2 (T-Cytoplasm) • Torres (S-Cytoplasm) n=8 Temperature regime • cold • warm • hot Illumination 16 h light (5000 Lux) 8 h darkness Standardized fertilisation and irrigation Temperature regime „hot" „warm" „cold" setting night/day a. sowing b. after 14 days c. after 21 days d. after 35 days a. sowing b. after 14 days c. after 21 days a. sowing b. after 14 days c. after 21 days 15°C / 20°C 15°C / 25°C 18°C / 28°C 22°C / 35°C 10°C / 15°C 15°C / 20°C 18°C / 25°C 21°C / 25°C 18°C / 25°C 16°C / 21°C Jobs Assessment of flowering characteristics Self-pollination – identification of pollen fertility (n = 3) Institute for Biosafety in Plant Biotechnology www.jki.bund.de Flowering Characteristic and Pollen Development At Main Flowering CMS maize hybrid Temperature Tassel regime hot DSP2 warm cold hot Torres warm cold 100% sterile 0% partly restored 0% fertile 0% sterile 50% partly restored 50% fertile 0% sterile 0% partly restored 100% fertile 100% sterile 0% partly restored 0% fertile 0% sterile 100% partly restored* 0% fertile 0% sterile 100% partly restored 0% fertile „hot“ DSP2 Pollen 100% no 0% few 0% many 0% no 0% few 100% many 0% no 0% few 100% many 100% no 0% few 0% many 0% no 100% few* 0% many 0% no 100% few 0% many „warm“ Torres „cold“ DSP2 * very weak/few Institute for Biosafety in Plant Biotechnology www.jki.bund.de Pollen fertility Number of kernels per ear 200 180 Kernels per ear 160 Temperature regime day/night 140 120 heiß 35°C/22°C 100 80 warm 25°C/18°C 60 40 kalt 21°C/16°C 20 0 DSP2 Torres Institute for Biosafety in Plant Biotechnology www.jki.bund.de 2nd Green House Experiment Flowering Characteristic and Pollen development CMS maize hybrid • DSP2 (T-cytoplasm) • Torres (S-cytoplasm) • Zidane (S-cytoplasm n=8 CMS maize hybrid hot DSP2 Temperature regime dito warm cold Illumination dito Jobs Assessment of flowering characteristics Self-pollination – identification of pollen fertility (n = 8) Temperature Tassel regime hot Torres warm cold hot Zidane warm cold Institute for Biosafety in Plant Biotechnology 100% sterile 0% partly restored 0% fertile 0% sterile 11% partly restored 89% fertile 0% sterile 0% partly restored 100% fertile 100% sterile 0% partly restored 0% fertile % sterile 100% partly restored 0% fertile 0% sterile 100% partly restored 0% fertile 0% sterile 100% partly restored 0% fertile 0% sterile 54% partly restored 46% fertile 0% sterile 60% partly restored 40% fertile Pollen 100% no 0% few 0% many 0% no 6% few 94% many 0% no 0% few 100% many 100% no 0% few 0% many 0% no 100% few * 0% many 0% no 100% few 0% many 100% no 0% few 0% many 0% no 97% few 3% many 0% no 70% few 30% many * very weak/few www.jki.bund.de Pollen fertility Number of kernels per ear 350 Kernels per ear 300 Temperature regime day/night 250 200 150 heiß 35°C/22°C 100 warm 25°C/18°C 50 kalt 21°C/16°C 0 DSP2 Torres Zidane Conclusions • high day and night temperatures support the sterility • as lower the day and night temperatures, as stronger the restoration of fertility • temperature effects clearly depending on the genotype Institute for Biosafety in Plant Biotechnology www.jki.bund.de Future prospects Introduction of GM-CMS maize hybrids in practice: • What amount of pollinator is necessary for a sufficient yield? • How should the pollinator be distributed within the field? Questions will be answered in the frame of the EU-funded project PRICE (Practical Implementation of Coexistence in Europe) Project no: FP7-KBBE-2011-5 Poster: Optimization of Spatial Arrangement of the Pollinator in Cytoplasmic Male Sterile (cms) Maize Grown as a Biological Confinement Method for Coexistence Heidrun Bückmann, Gemma Capellades, Kateřina Hamouzová, Josef Holec, Joaquima Messeguer, Anna Nadal, Maria Pla, Joan Serra, Josef Soukup, Katja Thiele and Joachim Schiemann Institute for Biosafety in Plant Biotechnology www.jki.bund.de Thank you very much for your attention! Institute for Biosafety in Plant Biotechnology www.jki.bund.de
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