, Volume 155, Issue 1–2, pp 143–152 | Cite as

Production of 2n pollen of Asiatic hybrid lilies by nitrous oxide treatment

  • Masako Akutsu
  • Satomi Kitamura
  • Ryo Toda
  • Ichiro Miyajima
  • Keiichi Okazaki


Tetraploid varieties of lilies have superior agronomic traits such as large flowers and resistance to physiological disorders. In the present study, we attempted to induce 2n pollen of Asiatic hybrid lilies by arresting the meiotic process with nitrous oxide (N2O) gas. To determine which meiotic stage is optimal for induction of 2n pollen, plants with attached buds at different meiotic stages were treated with N2O for 24 h in a pressure-tolerant cylinder. A few 2n pollen grains were induced using plants with anthers in prophase I, whereas mixed pollen grains of differing size were produced using plants undergoing meiotic metaphase predominantly in anthers. Although normal lily pollen grains are elliptical, nitrous oxide exposure induced giant pollen grains that appeared spherical. Flow cytometry analysis showed that the giant pollen grains were diploid. When mixed pollen that included normal and giant pollen was crossed to tetraploid cultivars, the resulting seedlings were tetraploid and aneuploid, indicating that the giant pollen grains were diploids that could generate tetraploid seedlings through fusion to diploid eggs supplied from a tetraploid female parent. Thus, treatment with N2O is useful for the production of 2n lily pollen and may provide a new approach for tetraploid lily breeding.


Chromosome doubling Flow cytometry Lilium Meiosis Nitrous oxide 2n pollen 



This work was supported in part by grants from the Ministry of Agriculture, Forestry and Fisheries, Japan and in part by a grant for the Promotion of Niigata University Research Projects.


  1. Arisumi T (1964) Colchicine-induced tetraploid and cytochimeral daylilies. J Hered 55:255–260Google Scholar
  2. Barba-Gonzalez R, Miller CT, Ramanna MS, Van Tuyl JM (2006) Nitrous oxide (N2O) induces 2n gametes in sterile F1 hybrids between Oriental × Asiatic lily (Lilium) hybrids and leads to intergenomic recombination. Euphytica 148:308–309CrossRefGoogle Scholar
  3. Berdahl JD, Barker RE (1991) Characterization of autotetraploid Russian wildrye produced with nitrous oxide. Crop Sci 31:1153–1155CrossRefGoogle Scholar
  4. Bretagnolle F, Thompson JD (1995) Gametes with the somatic chromosome number: mechanisms of their formation and role in the evolution of autopolyploid plants. New Phytol 129:1–22CrossRefGoogle Scholar
  5. Bullitta S, Scarpa GM, Smith RR, Veronesi F (1995) Unreduced gametes in ball clover and its relevance in white clover breeding. J Genet Breed 49:157–162Google Scholar
  6. Darlington CD, Hair JB, Hurcombe R (1951) The history of the garden hyacinths. Heredity 5:233–252Google Scholar
  7. DeGuzman R, Riggs D (2000) A survey of proteinases active during meiotic development. Planta 210:921–924PubMedCrossRefGoogle Scholar
  8. Den Nijs TPM, Peloquin SJ (1977) 2n gametes in potato species and their function in sexual polyploidization. Euphytica 26:585–600CrossRefGoogle Scholar
  9. Dvorak J, Harevery BL, Coulman BE (1973) The use of nitrous oxide for producing eupolyploids and aneuploids in wheat and barley. Can J Genet Cytol 15:205–214Google Scholar
  10. Emsweller SL (1949) Colchicine-induced polyploidy in Lilium longiflorum. Am J Bot 36:135–144CrossRefPubMedGoogle Scholar
  11. Emsweller SL, Uhring J (1960) Breeding Lilium longiflorum at the tetraploid level. Proc Am Soc Hortic Sci 75:711–719Google Scholar
  12. Filotico F, Carputo D, Barone A (1995) 2n pollen production in Solanum phurejaS. tuberosum hybrids. J Genet Breed 49:255–260Google Scholar
  13. Iizuka M, Ikeda A (1968) Induction of polyploidy in Lilium formosanum Wallace. Jpn J Genet 43:95–101Google Scholar
  14. Kato A (1997) An improved method for chromosome counting in maize. Biotech Histochem 72:249–252PubMedGoogle Scholar
  15. Kato A (2002) Chromosome doubling of haploid maize seedling using nitrous oxide gas at the flower primordial stage. Plant Breed 121:370–377CrossRefGoogle Scholar
  16. Ketsa S, Uthairatanakiji A, Prayurawong A (2001) Senescence of diploid and tetraploid cut inflorescences of Dendrobium ‘Caesar’. Sci Hortic 91:133–141CrossRefGoogle Scholar
  17. Kihara H, Nishiyama I (1932) Different compatibility in reciprocal crosses of Avena, with special reference to tetraploid hybrids between hexaploid and diploid species. Jpn J Bot 6:245–305Google Scholar
  18. Leslie AC (1982) The international lily register. 3rd edn. Royal Horticultural Society, LondonGoogle Scholar
  19. Lim KB, van der M Musisers JJM, van Tuyl JM (2002) Breeding for flower longevity enhancement of Asiatic hybrid lilies. Acta Hortic 570:409–413Google Scholar
  20. Maceira NO, De Haan AA, Lumaret R, Billon M, Delay J (1992) Production of 2n gametes in diploid subspecies of Dactylis glomerata L. 1. Occurrence and frequency of 2n pollen. Ann Bot 69:335–343Google Scholar
  21. McRae JF (1987) Stomatal size and pollen characteristics as an indication of chromosome numbers in lilies. Lily Yearb North Am Lily Soc 40:19–26Google Scholar
  22. Nishikawa T, Okazaki K, Arakawa K, Nagamine T (2001) Phylogenic analysis of section Sinomartagon in genus Lilium using sequences of the internal transcribed spacer region in nuclear ribosomal DNA. Breed Sci 51:39–46CrossRefGoogle Scholar
  23. Nygren A (1955) Polyploids in Melandrium produced by nitrous oxide. Hereditas 41:287–290Google Scholar
  24. Okazaki K, Hane Y (2005) Comparison of diploid and chimeric forms (4×/2×) of Asiatic hybrid lilies (Lilium spp.) under natural and early forcing culture. NZ J Crop Hortic Sci 33:261–267Google Scholar
  25. Okazaki K, Kurimoto K, Miyajima I, Enami A, Mizuochi H, Matsumoto Y, Ohya H (2005) Induction of 2n pollen in tulips by arresting meiotic process with nitrous oxide gas. Euphytica 143:101–114CrossRefGoogle Scholar
  26. Orjeda G, Freyre R, Iwanaga M (1990) Production of 2n pollen in diploid Ipomoea trifida, a putative wild ancestor of sweet potato. J Hered 81:462–467Google Scholar
  27. Östergren G (1954) Polyploids and aneuploids of Crepis capillaris produced by treatment with nitrous oxide. Genetica 24:54–64Google Scholar
  28. Östergren G (1957) Production of polyploids and aneuploids of Phalaris by means of nitrous oxide. Hereditas 43:512–516CrossRefGoogle Scholar
  29. Ramanna MS (1979) A re-examination of the mechanisms of 2n gametes formation in potato and its implications for breeding. Euphytica 28:537–561CrossRefGoogle Scholar
  30. Sano Y, Tanaka I (2005) A histone H3.3-like gene specifically expressed in the vegetative cell of developing lily pollen. Plant Cell Physiol 46:1299–1308PubMedCrossRefGoogle Scholar
  31. Takamura T, Miyajima I (1996) Colchicine induced tetraploids in yellow-flowered cyclamens and their characteristics. Sci Hortic 65:305–312CrossRefGoogle Scholar
  32. Taylor NL, Anderson MK, Quesenberry KH, Watson L (1976) Doubling the chromosome number of Trifolium species using nitrous oxide. Crop Sci 16:516–518CrossRefGoogle Scholar
  33. Van Santen E, Hugessen PM, Casler MD (1991) Identification and frequency of tetraploid progeny from 2x–4x and 4x–2x crosses in Dactylis. Genome 34:273–278Google Scholar
  34. Van Tuyl JM (1990) Research on mitotic and meiotic polyploidization in lily breeding. Herbertia 45:97–103Google Scholar
  35. Van Tuyl JM, De Vries JM, Bino RJ, Kwakkenbos TAM (1989) Identification of 2n-pollen producing interspecific hybrids of Lilium by flow cytometry. Cytologia 54:737–745Google Scholar
  36. Van Tuyl JM, Meijer B, van Diën MP (1992) The use of oryzalin as an alternative for colchicine in in-vitro chromosome doubling of Lilium and Nerine. Acta Hortic 352:625–630Google Scholar
  37. Veronesi F, Mariani A, Bingham ET (1986) Unreduced gametes in diploid Medicago and their importance in alfalfa breeding. Theor Appl Genet 72:37–41CrossRefGoogle Scholar
  38. Wylie AP (1952) The history of the garden narcissi. Heredity 6:137–156Google Scholar
  39. Zeilinga AE, Schouten HP (1968) Polyploidy in garden tulips. II. The production of tetraploids. Euphytica 17:303–310Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Masako Akutsu
    • 1
    • 3
  • Satomi Kitamura
    • 1
  • Ryo Toda
    • 1
  • Ichiro Miyajima
    • 2
  • Keiichi Okazaki
    • 1
  1. 1.Faculty of AgricultureNiigata UniversityIkarashiJapan
  2. 2.Horticultural Research CenterNiigata Agricultural Research InstituteSeiroJapan
  3. 3.Local Crop Breeding Research TeamNational Agricultural Research Center for Hokkaido RegionSapporoJapan

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