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Somatic embryogenesis and plant regeneration of tropical maize genotypes

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Abstract

In Latin America and sub-Saharan Africa, tropical maize (Zea mays L.) is a major crop for human consumption. To cope with the increasing population and changing environment, there is a need for improving tropical maize germplasm. As part of a biotechnological approach, efficient in vitro regeneration of two tropical maize inbred lines (CML216 and CML244) was established. A number of parameters were optimized, such as age of the immature embryos, plant media and growth regulator concentration. After 6 weeks of culture, somatic embryos that had already reached the coleoptilar stage produced shoots after light induction and developed into fertile plants after acclimation in the soil. The callus induction frequencies and somatic embryo-derived plantlet formation were higher when cultured with the Linsmaier and Skoog medium than those with the Chu’s N6 basal medium. Regeneration of tropical maize shoots depended on the 2,4-dichlorophenoxyacetic acid (2,4-D) concentration at the callus initiation stage from immature embryos. The recalcitrance of the tropical maize inbred line TL26 to in vitro regeneration was overcome in a single-cross hybrid with the CML216 and CML244 genotypes. Remarkably, tropical maize somatic embryos were formed at the abaxial side of the scutellum facing the medium, probably from the axis of the immature embryos, as shown by histological sections. Upon co-cultivation, agrobacteria transiently expressed their intronless β-glucuronidase-encoding gene at the embryogenic tissue, but not with an intron-containing gene, suggesting that virulence genes are induced in Agrobacterium, but that subsequent steps in the T-DNA transfer are inhibited.

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Abbreviations

2,4-D:

2,4-Dichlorophenoxyacetic acid

bar :

Phosphinothricin acetyltransferase-encoding gene

CIM:

Callus induction medium or resting medium

CMM:

Callus maturation medium

ESM/Inf:

Embryo suspension medium/infection medium

GUS:

β-glucuronidase

LS:

Linsmaier and Skoog

MES:

2-(N-morpholino)ethanesulphonic acid

MS:

Murashige and Skoog

N6:

Chu’s N6 basal medium

P35:

Cauliflower mosaic virus 35S promoter

PIPES:

Piperazine-N,N-bis(2-ethanesulphonic acid)

R0:

Regenerated shoot from somatic embryo

R1:

Progeny of R0 shoot

SIM:

Shoot induction medium or regeneration medium

T-DNA:

Transfer DNA

T35S:

Cauliflower mosaic virus 35S terminator

Tnos:

Nopaline synthase terminator

UBIL:

Maize long ubiquitin promoter

uidA :

β-d-glucuronidase gene from Escherichia coli

X-Gluc:

5-Bromo-4-chloro-3-indolyl-β-d-glucuronic acid

YEP:

Yeast Extract Peptone

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Acknowledgments

We thank Dr. Martine De Cock for help in preparing the manuscript. S.E.A., A.J.M., and C.T. are indebted to the Vlaamse Interuniversitaire Raad, The Rockefeller Foundation, and the United States Agency for International Development for predoctoral fellowships, respectively.

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Authors and Affiliations

  1. Plant Transformation Facility, Department of Biochemistry and Biotechnology, Kenyatta University, P.O. Box 43844, Nairobi, Kenya

    Sylvester Elikana Anami, Allan Jalemba Mgutu & Jesse Machuka

  2. Department of Plant Systems Biology, VIB, 9052, Gent, Belgium

    Sylvester Elikana Anami, Griet Coussens, Mansour Karimi, Pierre Hilson & Mieke Van Lijsebettens

  3. Department of Plant Biotechnology and Genetics, Ghent University, 9052, Gent, Belgium

    Sylvester Elikana Anami, Griet Coussens, Mansour Karimi, Pierre Hilson & Mieke Van Lijsebettens

  4. Kenya Agricultural Research Institute, P.O.Box 57811, Nairobi, Kenya

    Catherine Taracha

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  1. Sylvester Elikana Anami
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  2. Allan Jalemba Mgutu
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  3. Catherine Taracha
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Correspondence to Mieke Van Lijsebettens.

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Anami, S.E., Mgutu, A.J., Taracha, C. et al. Somatic embryogenesis and plant regeneration of tropical maize genotypes. Plant Cell Tiss Organ Cult 102, 285–295 (2010). https://doi.org/10.1007/s11240-010-9731-7

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  • DOI: https://doi.org/10.1007/s11240-010-9731-7

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