Advertisement

Generation of Interspecific Hybrids of Trifolium Using Embryo Rescue Techniques

  • Ajoy Kumar Roy
  • Devendra Ram Malaviya
  • Pankaj Kaushal
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 710)

Abstract

The genus Trifolium Leguminosae (Fabaceae), commonly called clovers, includes 237–290 annual and perennial species, of which about 20 are important as cultivated and pasture crops. Taxonomic distribution supported by molecular analysis indicates that Mediterranean region is one of the main centers of distribution of the genus and also a center of domestication and breeding. Self-incompatibility is prevalent in the genus, controlled by a single, multiallelic gene expressed gametophytically in the pollen. It was suggested that hybridity did not play a major role in the evolution of the genus due to the poor crossability of the species under natural conditions. Interspecific hybridization in the genus Trifolium by conventional crossing techniques has been largely unsuccessful. Post-zygotic barriers appear to be a primary cause of the reproductive isolation, associated with endosperm disintegration and consequent abnormal differentiation and starvation of the hybrid embryo. As hybridization using conventional techniques has almost failed in Trifolium, embryo culture technique was used by breeders to obtain new combinations of interspecific hybrids. Embryo culture has been effectively used in developing interspecific hybrids in Trifolium ambiguum, T. pratense, T. montanum, T. occidentale, T. isthomocarpum, T. repens, T. nigrescens, T. uniflorum, T. sarosiense, T. alexandrinum, T. apertum, T. resupinatum, T. constantinopolitanum, T. rubens, and T. alpestre in various combinations. The successful embryo ­rescue and development of hybrid plantlets requires skilled techniques of tissue culture and field practices. It includes hybridization in field; excision of hybrid embryos at appropriate stage; disinfection and culture in suitable culture media to allow maturation of embryo, multiplication of shoots, and rooting; hardening of the plantlets; inoculation with suitable Rhizobium culture; and transfer to field.

Key words

Clovers Embryo rescue Forage Interspecific hybridization Pasture Tissue culture Trifolium Zygote 

References

  1. 1.
    Gillett JM (1952) The genus Trifolium in southern Arabia and in Africa south of Sahara. Kew Bull 7:367–404CrossRefGoogle Scholar
  2. 2.
    Watson LE, Sayed-Ahmed H, Badr A (2000) Molecular phylogeny of old world Trifolium (Fabaceae), based on plastid and nuclear markers. Plant Syst Evol 224:153–171CrossRefGoogle Scholar
  3. 3.
    Ellison NW, Liston A, Steiner JJ, Williams WM, Taylor NL (2006) Molecular phylogenetics of the clover genus (Trifolium – Leguminosae). Mol Phylogenet Evol 39:688–705PubMedCrossRefGoogle Scholar
  4. 4.
    Zohary M (1972) Origin and evolution in the genus Trifolium. Bot Not 125:501–511Google Scholar
  5. 5.
    Zohary M, Heller D (1984) The genus Trifolium. The Israel Academy of Sciences and Humanities, JerusalemGoogle Scholar
  6. 6.
    Atwood SS (1940) Genetics of cross-­incompatibility among self-incompatible plants of Trifolium repens. J Am Soc Agron 32:955–968CrossRefGoogle Scholar
  7. 7.
    Williams W (1951) Genetics of incompatibility in alsike clover, Trifolium hybridum. Heredity 5:51–73PubMedCrossRefGoogle Scholar
  8. 8.
    Atwood SS (1944) Oppositional alleles in natural populations of Trifolium repens. Genetics 29:428–435PubMedGoogle Scholar
  9. 9.
    Lawrence MJ (1996) Number of incompatibility alleles in clover and other species. Heredity 76:610–615CrossRefGoogle Scholar
  10. 10.
    Roy AK, Malaviya DR, Kaushal P (2005) Pollination behaviour in different breeding populations in Egyptian clover. Plant Breed 124:171–175CrossRefGoogle Scholar
  11. 11.
    Wexelsen H (1928) Chromosome numbers and morphology in Trifolium. Calif Univ Agric Sci 2:355–376Google Scholar
  12. 12.
    Malaviya DR, Roy AK, Kaushal P, Kumar B, Tewari A (2008) Genetic similarity among Trifolium species based on isozyme banding pattern. Plant Syst Evol 276:125–136CrossRefGoogle Scholar
  13. 13.
    Marshall AH, Michaelson-Yeats TPT, Aluka P, Meredith M (1995) Reproductive characters of interspecific hybrids between Trifolium repens L. and T. nigrescens Viv. Heredity 74:136–145CrossRefGoogle Scholar
  14. 14.
    Gibson PB, Chen CC, Gillingham JT, Barnett OW (1971) Interspecific hybridization of Trifolium uniflorum L. Crop Sci 11:895–899CrossRefGoogle Scholar
  15. 15.
    Williams E (1978) A hybrid between Trifolium repens and T. ambiguum obtained with the aid of embryo culture. N Z J Bot 16:499–506Google Scholar
  16. 16.
    Chen CC, Gibson PR (1971) Seed development following the mating of Trifolium repens  ×  T. uniflorum. Crop Sci 11:667–672CrossRefGoogle Scholar
  17. 17.
    White DWR, Williams E (1976) Early seed development after crossing of Trifolium semiplosum and T. repens. N Z J Bot 14:161–168Google Scholar
  18. 18.
    Williams E, White DWR (1976) Early seed development after crossing of Trifolium ambiguum and T. repens. In: Proceedings of the 3rd international congress of SABRAO, vol 2, pp 26–30Google Scholar
  19. 19.
    Kazimierska EM (1978) Embryological studies of cross compatibility in the genus Trifolium L. II Fertilization, development of embryo and endosperm in crossing T. repens L. with T. medium L. Genet Pol 19:15–24Google Scholar
  20. 20.
    Keim WF (1953) Interspecific hybridization in Trifolium utilizing embryo culture techniques. Agron J 45:601–606CrossRefGoogle Scholar
  21. 21.
    Phillips GC, Grosser JW, Berger S, Taylor NL, Collins GB (1992) Interspecific hybridization between red clover and Trifolium alpestre using in vitro embryo rescue. Crop Sci 32:1113–1115CrossRefGoogle Scholar
  22. 22.
    Ferguson NH, Rupert EA, Evans PT (1990) Interspecific Trifolium hybrids produced by embryo and ovule culture. Crop Sci 30:1145–1149CrossRefGoogle Scholar
  23. 23.
    Collins GB, Taylor NL, Phillips GC (1981) Successful hybridization of red clover with perennial Trifolium species via embryo rescue. In: Smith TA, Hays VW (eds) International grassland congress, Lexington, vol 14. Westview, Boulder, CO, pp 168–170Google Scholar
  24. 24.
    Pandey KK, Grant JE, Williams EG (1987) Interspecific hybridization between Trifolium. Aust J Bot 35:171–182CrossRefGoogle Scholar
  25. 25.
    Yamada T, Fukuoka H (1986) Production of interspecific hybrids between Trifolium ambiguum M. Bieb. and T. repens L. by ovule culture. Jpn J Breed 36:233–239Google Scholar
  26. 26.
    Przywara L, White DWR, Sanders PM, Maher D (1989) Interspecific hybridization of Trifolium repens with T. hybridum using in ovulo embryo and embryo culture. Ann Bot 64:613–624Google Scholar
  27. 27.
    Sawai A, Ueda S, Gau M, Uchiyama K (1990) Interspecific hybrids of Trifolium medium L.  ×  4× T. pratense L. obtained through embryo culture. J Jpn Soc Grassl Sci 35:267–272Google Scholar
  28. 28.
    Sawai A, Yamaguchi H, Uchiyama K (1995) Fertility and morphology of the chromosome-doubled hybrid Trifolium medium  ×  T. pratense (red clover) and backcross progeny. Grassl Sci 41:122–127Google Scholar
  29. 29.
    Roy AK, Malaviya DR, Kaushal P, Kumar B, Tiwari A (2004) Interspecific hybridization of T. alexandrinum with T. constantinopolitanum using embryo rescue. Plant Cell Rep 22:605–610CrossRefGoogle Scholar
  30. 30.
    Malaviya DR, Roy AK, Kaushal P, Kumar B, Tiwari A (2004) Development and characterization of interspecific hybrids of Trifolium alexandrinum  ×  T. apertum using embryo rescue. Plant Breed 123:536–542CrossRefGoogle Scholar
  31. 31.
    Kaushal P, Malaviya DR, Roy AK, Kumar B, Tiwari A (2005) Trifolium alexandrinum  ×  T. resupinatum – interspecific hybrids developed through embryo rescue. Plant Cell Tissue Organ Cult 83:137–144CrossRefGoogle Scholar
  32. 32.
    Selim AK, Abdel-Tawab FM, Fahmy EM (1977) Phylogenetic relationship in genus Trifolium L. I. Interspecific crossability and serological affinities. Egypt J Genet Cytol 6:274–283Google Scholar
  33. 33.
    Trimble JP, Hovin AW (1960) Interspecific hybridization of certain Trifolium species. Agron J 52:405CrossRefGoogle Scholar
  34. 34.
    Evans AM (1962) Species hybridization in Trifolium. II. Investigating the pre-fertilization barriers to compatibility. Euphytica 11:256–262CrossRefGoogle Scholar
  35. 35.
    Rupert EA, Evans PT (1980) Embryo development after interspecific cross-pollinations among species of Trifolium, section Lotoidea, Agronomy abstracts. American Society of Agronomy, Madison, WI, p 68Google Scholar
  36. 36.
    Phillips GC, Collins GB, Taylor NL (1982) Interspecific hybridization of red clover (Trifolium pratense) with T. sarosiense using in vitro embryo rescue. Theor Appl Genet 62:17–24Google Scholar
  37. 37.
    Williams EG, De Lautour G (1980) The use of embryo culture with transplanted nurse endosperm for the production of interspecific hybrids in pasture legumes. Bot Gaz 141:252–257CrossRefGoogle Scholar
  38. 38.
    Yamada T, Fukoka H (1985) Application of ovule culture to interspecific hybridization between Trifolium repens and T. ambiguum. In: Proceedings of the XVth International Grassland Congress, pp 241–243. held at Kyoto August 24–31, 1985 Published by The Science Council of Japan. The Japanese Society of Grassland Science.Google Scholar
  39. 39.
    Nakajima T, Doyama Y, Matsumoto H (1969) In vitro culture of excised ovules of white clover Trifolium repens L. Jpn J Breed 19:373–378Google Scholar
  40. 40.
    Brewbaker JL, Keim WF (1953) A fertile interspecific hybrid in Trifolium. Am Nat 8:323–326CrossRefGoogle Scholar
  41. 41.
    Pandey K (1957) A self compatible species in Trifolium. J Hered 48:278–281Google Scholar
  42. 42.
    Kazimierski T, Kazimierska EM (1968) Investigations of hybrids of the genus Trifolium L., I sterile hybrid T. repens  ×  T. xerocephalum (in Polish). Acta Soc Bot Pol 37:549–560Google Scholar
  43. 43.
    Gibson PB, Beinhart G (1969) Hybridization of Trifolium occidantale with two other species of clover. J Hered 60:93–96Google Scholar
  44. 44.
    Kazimierski T, Kazimierska EM (1973) Investigations of hybrids of the genus Trifolium L., IV. Cytogenetics of the cross T. repens  ×  T. isthmocarpum Brot. (in Polish). Acta Soc Bot Pol 41:127–147Google Scholar
  45. 45.
    Williams E, Verry IM (1981) A partially fertile hybrid between Trifolium repens and T. ambiguum. N Z J Bot 19:1–7Google Scholar
  46. 46.
    Armstrong KC, Cleveland RW (1970) Hybrids of Trifolium pratense  ×  Trifolium ­pallidum. Crop Sci 10:354–357CrossRefGoogle Scholar
  47. 47.
    Nitsch JP (1951) Growth and development in vitro of excised ovaries. Am J Bot 38:566–577CrossRefGoogle Scholar
  48. 48.
    Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  49. 49.
    Phillips GC, Collins GB (1984) Red clover and other forage legumes. In: Sharp WR, Evans DA, Ammirato PV, Yamada Y (eds) Handbook of plant cell culture, vol 2. Macmillan, New York, pp 169–210Google Scholar

Copyright information

© Humana Press 2011

Authors and Affiliations

  • Ajoy Kumar Roy
    • 1
  • Devendra Ram Malaviya
    • 1
  • Pankaj Kaushal
    • 2
  1. 1.Indian Grassland and Fodder Research InstituteJhansiIndia
  2. 2.Central Rice Research InstituteCuttackIndia

Personalised recommendations