Poorly formed chloroplasts are barriers to successful interspecific hybridization in chickpea following in vitro embryo rescue

  • Heather J. Clarke
  • Maya Kumari
  • Tanveer N. Khan
  • Kadambot H. M. Siddique
Original Paper


Albinism is viewed as a major experimental bottleneck during wide hybridization in several species; the phenomenon is also widely reported in androgenesis and doubled haploid cultures. In this study, when chickpea (Cicer arietinum) was crossed with distant wild relatives, C. judaicum and C. pinnatifidum, the only hybrid embryos that survived were those rescued using in vitro techniques. Fourteen to 21-day-old embryos were incubated in ML6 medium with 90 g l−1 sucrose, 1 mg l−1 zeatin and 0.25 mg l−1 indole acetic acid. Germinating embryos were dissected from the ovule and transferred to fresh medium without growth regulators. Later, shoots were micropropagated on solid MS medium, supplemented with B5 vitamins, 30 g l−1 sucrose, 0.1 mg l−1 6-benzyladenine and 0.01 mg l−1 naphthaleneacetic acid. Although some post-hybridization barriers were overcome by rescue in vitro, regenerated hybrid plantlets failed to thrive in culture and few survived transfer to soil. Here we report findings from characterization of this valuable breeding material, as a step towards a better understanding of albinism in chickpea wide hybrids and other plant tissue cultures. Following proliferation, hybrids were phenotyped as green, pale green and albino. Genotype affected pod set, regeneration and albinism. Plastid ultrastructure studies using transmission electron microscopy revealed that thylakoid membranes were well-formed in green hybrids but not in albinos. Spectrophotometric analysis of chlorophyll a, chlorophyll b and total carotenoids revealed that chlorophyll content was three to fourfold lower in albino compared to green hybrids; moreover green hybrids had two to threefold lower total chlorophyll content than in vitro-grown plantlets of their parents. In contrast, carotenoids were higher in some albino phenotypes and lower in others; however overall differences in carotenoids were less marked between all genotypes compared to chlorophyll pigments. Genetic variability between different wide crosses provides an opportunity to select certain chickpea parents and wild species which give rise to more frequent green hybrid regenerants. In future, only these hybrids will be maintained and multiplied for transfer to the glasshouse in our program.


Cicer wide hybrids Embryo rescue Pigments Plastid Ultrastructure 



We thank Professor John Kuo for his guidance and Ms Lyn Krilak for technical assistance in the Centre for Microscopy Characterization and Analysis at The University of Western Australia (UWA). We also acknowledge valuable suggestions of Prof. H.K Lichtenthaler, University of Karlsruhe, Germany, regarding chlorophyll pigment analysis. The Grains Research and Development Corporation (Project UWA00091) provided support to develop tissue culture technology and a Cicer interspecific breeding program. The Department of Education, Science and Training, Australia supported the visit of Dr Maya Kumari from India to Australia through an Endeavour Research Fellowship for postdoctoral research.


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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Heather J. Clarke
    • 1
    • 2
  • Maya Kumari
    • 1
    • 3
  • Tanveer N. Khan
    • 1
    • 2
  • Kadambot H. M. Siddique
    • 1
    • 2
    • 4
  1. 1.Centre for Legumes in Mediterranean Agriculture, Faculty of Natural and Agricultural SciencesThe University of Western AustraliaCrawleyAustralia
  2. 2.The UWA Institute of AgricultureThe University of Western AustraliaCrawleyAustralia
  3. 3.Defence Institute of Bio-Energy Research (DIBER)Defence Research and Development OrganisationGoraparao, HaldwaniIndia
  4. 4.College of Food and Agricultural SciencesKing Saud UniversityRiyadhSaudi Arabia

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