Sexual Plant Reproduction

, Volume 23, Issue 4, pp 255–264 | Cite as

An uncoupling screen for autonomous embryo mutants in Arabidopsis thaliana

  • Nick Fenby
  • Hong Pu
  • Roger Pennell
  • Uta Praekelt
  • Rob Day
  • Rod ScottEmail author
Technical Advance


Simple de novo screens in Arabidopsis thaliana have previously identified mutants that affect endosperm development but viable-embryo mutants have not been identified. Our strategy to identify autonomous embryo development was to uncouple embryo and endosperm fertilisation. This involved a male-sterile mutant population being crossed with a distinct pollen parent—the pollen was needed to initiate endosperm development and because it was distinct, the maternal progeny could be selected from the hybrid population. This process was refined over three stages, resulting in a viable approach to screen for autonomous embryo mutants. From 8,000 screened plants, a mutation was isolated in which the integument cells extended from the ovule and proliferated into a second complete twinned ovule. Some embryos from the mutant were normal but others developed fused cotyledons. In addition, a proportion of the progeny lacked paternal genes.


Apomixis Embryo Fused cotyledons Mutant screening Twin ovule 



We thank D. Hird and R. Hodge for technical advice and R. Vinkenoog for advice in manuscript preparation. M.-A. Van Sluys is thanked for providing TZ sequence prior to publication. We are grateful to BBSRC for funding UP and NF, to Ceres Inc. for funding NF and Sulis Innovations who funded NF and RD. We are grateful to ABRC and NASC for the supply of seeds.


  1. Adams S, Vinkenoog R, Spielman M, Dickinson HG, Scott RJ (2000) Parent-of-origin effects on seed development in Arabidopsis thaliana require DNA methylation. Development 127:2493–2502PubMedGoogle Scholar
  2. Asker S, Jerling L (1992) Apomixis in plants. CRC Press, Boca RatonGoogle Scholar
  3. Bailey JP, Stace CA (1992) Chromosome number, morphology, pairing, and DNA values of species and hybrids in the genus Fallopia (Polygonaceae). Pl Systemat Evol 180:29–52CrossRefGoogle Scholar
  4. Baker SC, Robinson-Beers K, Villanueva JM, Gaiser JC, Gasser CS (1997) Interactions among genes regulating ovule development in Arabidopsis thaliana. Genetics 145:1109–1124PubMedGoogle Scholar
  5. Berthaud J (2001) Apomixis and the management of genetic diversity. In: Savidan Y, Carman JG, Dresselhaus T (eds) Flowering of Apomixis: from mechanisms to genetic engineering. CIMMYT Publications, Houston, pp 8–23Google Scholar
  6. Bushell C, Spielman M, Scott RJ (2003) The basis of natural and artificial postzygotic hybridisation barriers in Arabidopsis species. Plant Cell 15:1–14CrossRefGoogle Scholar
  7. Chaudhury AM, Ming L, Miller C, Craig S, Dennis ES, Peacock WJ (1997) Fertilisation-independent seed formation in Arabidopsis thaliana. Proc Natl Acad Sci (USA) 94:4223–4228CrossRefGoogle Scholar
  8. Dujardin M, Hanna WW (1989) Crossability of pearl millet with wild Pennisetum species. Crop Sci 29:77–80CrossRefGoogle Scholar
  9. Edwards K, Johnstone C, Thompson C (1991) A simple and rapid method for the preparation of genomic DNA for PCR analysis. Nucleic Acids Res 19:1349CrossRefPubMedGoogle Scholar
  10. Elliott RC, Betzner AS, Huttner F, Oakes MP, Tucker WQJ et al (1996) AINTEGUMENTA an APETALA2-like gene of Arabidopsis with pleiotropic roles in ovule development and floral organ growth. Plant Cell 8:155–168CrossRefPubMedGoogle Scholar
  11. Gaiser JC, Robinson-Beers K, Gasser CS (1995) The Arabidopsis SUPERMAN gene mediates asymmetric growth of the outer integuments of ovules. Plant Cell 7:333–345CrossRefPubMedGoogle Scholar
  12. Geldner N, Richter S, Vieten A, Marquardt S, Torrez-Ruiz RA, Mayer U, Jurgens G (2004) Partial loss-of-function alleles reveal a role for GNOM in auxin transport-related, post-embryogenic development of Arabidopsis. Development 131:389–400CrossRefPubMedGoogle Scholar
  13. Grossniklaus U, Vielle Calzada J-P, Hoepper M, Gagliana W (1998) Maternal control of embryogenesis by MEDEA, a polycomb-group gene in Arabidopsis. Science 280:446–450CrossRefPubMedGoogle Scholar
  14. Guerineau F, Sorensen A-M, Fenby N, Scott RJ (2003) Temperature sensitive diphtheria toxin confers conditional male-sterility in Arabidopsis thaliana. Plant Biotechnol 1:33–42CrossRefGoogle Scholar
  15. Guitton A-E, Berger F (2005) Loss of function of MULTICOPY SUPPRESSOR OF IRA 1 produces nonviable parthenogenetic embryos in Arabidopsis. Curr Biol 15:750–754CrossRefPubMedGoogle Scholar
  16. Guitton A-E, Page DR, Chambrier P, Lionner C, Faure J-E, Grossniklaus U, Berger F (2004) Identification of new members of fertilisation independent seed polycomb group pathway involved in the control of seed development in Arabidopsis thaliana. Development 131:2971–2981CrossRefPubMedGoogle Scholar
  17. Hanna WW, Powell JB (1973) Stubby head, an induced facultative apomict in pearl millet. Crop Sci 13:726–728CrossRefGoogle Scholar
  18. Hardtke CS, Berleth T (1998) The Arabidopsis gene MONOPTEROS encodes a transcription factor mediating embryo axis formation and vascular development. EMBO 17:1405–1411CrossRefGoogle Scholar
  19. Hecht V, Vielle-Calzada JP, Hartog MV, Schmidt EDL, Boutilier K, Grossniklaus U, de Vries SC (2001) The Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1 gene is expressed in developing ovules and embryos and enhances embryogenic competence in culture. Plant Physiol 177:803–816CrossRefGoogle Scholar
  20. Hennig L, Taranto P, Walser M, Schönrock N, Gruissem W (2003) Arabidopsis MSI1 is required for epigenetic maintenance of reproductive development. Development 130:2555–2565CrossRefPubMedGoogle Scholar
  21. Klucher KM, Chow H, Reiser L, Fischer RL (1996) The AINTEGUMENTA gene of Arabidopsis required for ovule and female gametophyte development is related to the floral homeotic gene APETALA2. Plant Cell 8:137–153CrossRefPubMedGoogle Scholar
  22. Leblanc O, Grimanelli D, Gonzáles de León D, Savidan Y (1995) Detection of the apomictic mode of reproduction in maize-Tripsacum hybrids using maize RFLP markers. Theor Appl Genet 90:1198–1203CrossRefGoogle Scholar
  23. Leon-Kloosterziel KM, Keijzer CJ, Koornneef M (1994) A seed shape mutant of Arabidopsis that is affected in integument development. Plant Cell 6:385–392CrossRefPubMedGoogle Scholar
  24. Lotan T, Ohto M, Yee MK, West MA, Lo R, Kwong RW, Yamagishi K, Fischer RL, Goldberg RB, Harada JJ (1998) Arabidopsis LEAFY COTYLEDON 1 is sufficient to induce embryo development in vegetative tissue. Cell 93:1195–1205CrossRefPubMedGoogle Scholar
  25. Lui C, Xu Z, Chua N-H (1993) Auxin polar transport is essential for the establishment of bilateral symmetry during early plant embryogenesis. Plant Cell 5:621–630Google Scholar
  26. Mayer U, Buttner G, Jurgens G (1993) Apical-basal pattern formation in Arabidopsis embryo: studies on the role of the GNOM gene. Development 117:149–162Google Scholar
  27. McAbee JM, Hill TA, Skinner DJ, Izhakr A, Hauser BA, Meister RJ, Reddy GV, Meyerowitz EM, Bowman JL, Gasser CS (2006) ABBERANT TESTA SHAPE encodes a KANADI family member, linking polarity determination to separation and growth of Arabidopsis ovule integuments. Plant J 46:522–531CrossRefPubMedGoogle Scholar
  28. Meinke DW, Franzmann LH, Nickle TC, Yeung EC (1994) Leafy cotyledon mutants of Arabidopsis. Plant Cell 6:1049–1064CrossRefPubMedGoogle Scholar
  29. Morgan RN, Alvernaz J, Aurthur L, Hanna WW, Ozias-Akins P (1997) Genetic characterisation and floral development of female sterile and stubby head, two aposporous mutants of pearl millet. Sex Plant Reprod 10:127–135CrossRefGoogle Scholar
  30. Naumova TN, Vielle-Calzada JP (2001) Ultrastructural analysis of apomictic development. In: Savidan Y, Carman JG, Dresselhaus T (eds) Flowering of Apomixis: from mechanisms to genetic engineering. CIMMYT Publications, Houston, pp 212–228Google Scholar
  31. Naumova TN, Van Der Laak J, Osadtchiy J, Matzk F, Kravtchenko A, Bergervoet J, Ramulu KS, Boutilier K (2001) Reproductive development in apomictic populations of Arabis holboellii (Brassicaceae). Sex Plant Reprod 14:195–200CrossRefGoogle Scholar
  32. Ogas J, Cheng J-C, Sung RZ, Somerville C (1997) Cellular differentiation regulated by gibberelin in the Arabidopsis thaliana pickle mutant. Science 277:91–94CrossRefPubMedGoogle Scholar
  33. Ogas J, Kaufmann S, Henderson J, Somerville C (1999) PICKLE is a CHD3 chromatin-remodeling factor that regulates the transition from embryogenic to vegetative development in Arabidopsis. Proc Natl Acad Sci USA 96:5319–5324CrossRefGoogle Scholar
  34. Ohad N, Margossian L, Hsu Y-C, Williams C, Repetti P, Fischer RL (1996) A mutation that allows endosperm development without fertilisation. Proc Natl Acad Sci USA 93:5319–5324CrossRefPubMedGoogle Scholar
  35. Okada K, Ueda J, Komaki MK, Bell CJ, Shimura Y (1991) Requirements of the auxin polar transport system in early stages of Arabidopsis floral bud formation. Plant Cell 3:677–684CrossRefPubMedGoogle Scholar
  36. Ozias-Akins P, Lubbers EL, Hanna WW, McNay JW (1993) Transmission of the apomictic mode of reproduction in Pennisetum: co-inheritance of the trait and molecular markers. Theor Appl Genet 85:632–638CrossRefGoogle Scholar
  37. Praekelt U, Scott R (2001) Induction of apomixis in sexual plants by apomixis. In: Savidan Y, Carman JG, Dresselhaus T (eds) Flowering of Apomixis: from mechanisms to genetic engineering. CIMMYT Publications, Houston, pp 212–228Google Scholar
  38. Ravi M, Marimuthu MPA, Siddiqi I (2008) Gamete formation without meiosis in Arabidopsis. Nature 451:1121–1124CrossRefPubMedGoogle Scholar
  39. Redei GP, Koncz C (1992) Classical mutagenesis. In: Koncz C, Chua NH, Schell J (eds) Methods in Arabidopsis research. World Scientific, London, pp 16–82Google Scholar
  40. Robinson-Beers K, Pruitt RE, Gasser CS (1992) Ovule development in wild-type Arabidopsis and two female sterile mutants. Plant Cell 4:1237–1249CrossRefPubMedGoogle Scholar
  41. Savidan Y (2001) Transfer of apomixis through wide crosses. In: Savidan Y, Carman JG, Dresselhaus T (eds) Flowering of Apomixis: from mechanisms to genetic engineering. CIMMYT Publications, Houston, pp 212–228Google Scholar
  42. Schmidt ED, Guzzo F, Toonen MA, de Vries SC (1997) A leucine–rich repeat containing receptor-like kinase marks somatic plant cells competent to form embryos. Development 124:2049–2062PubMedGoogle Scholar
  43. Schranz ME, Dobes C, Koch MA, Mitchell-Olds T (2005) Sexual reproduction, hybridization, apomixis, and polyploidization in the genus Boechera (Brassicaceae). Am J Bot 92:1797–1810CrossRefGoogle Scholar
  44. Schruff MC, Spielman M, Tiwari S, Adams S, Fenby N, Scott RJ (2006) The megaintegumenta/auxin response factor 2 gene of Arabidopsis links auxin signalling, cell division, and size of seeds and other organs. Development 133:251–261CrossRefPubMedGoogle Scholar
  45. Sorensen A, Guerineau F, Canales-Holzeis C, Dickinson HG, Scott RJ (2002) A novel extinction screen in Arabidopsis thaliana identifies mutant plants defective in early microsporangial development. Plant J 29:581–594CrossRefPubMedGoogle Scholar
  46. Stone SL, Kwong LW, Yee KM, Pelletier J, Lepiniec L, Fischer RL, Goldberg RB, Harada JJ (2001) LEAFY COTYLEDON2 encodes a B3 domain transcription factor that induces embryo development. Proc Natl Acad Sci USA 98:11806–11811CrossRefPubMedGoogle Scholar
  47. Taskin KM, Turgut K, Scott RJ (2004) Apomictic development in Arabis gunnisoniana. Isr J Plant Sci 52:155–160CrossRefGoogle Scholar
  48. West MAL, Yee KM, Danao J, Zimmerman JL, Fischer RL, Goldberg RB, Harada JJ (1994) LEAFY COTYLEDON1 is an essential regulator of late embryogenesis and cotyledon identity in Arabidopsis. Plant Cell 6:1731–1745CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Nick Fenby
    • 1
  • Hong Pu
    • 2
  • Roger Pennell
    • 2
  • Uta Praekelt
    • 3
    • 4
  • Rob Day
    • 1
    • 5
  • Rod Scott
    • 1
    Email author
  1. 1.Department of Biology and BiochemistryBath UniversityBathUK
  2. 2.Ceres Inc.Thousand OaksUSA
  3. 3.Department of BiologyLeicester UniversityLeicesterUK
  4. 4.Department of GeneticsLeicester UniversityLeicesterUK
  5. 5.Department of BiochemistryUniversity of OtagoDunedinNew Zealand

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