Transformation and Regeneration Protocol for Two Farmer Preferred Open Pollinated Tropical Maize (<i>Zea Mays</i>) Varieties

Johnstone Omukhulu Neondo; Amos Emitati Alakonya; Jonathan Matheka; Joel Okoyo Masanga; Remmy Wekesa Kasili

doi:doi:10.11648/j.jps.20160404.14

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Transformation and Regeneration Protocol for Two Farmer Preferred Open Pollinated Tropical Maize (Zea Mays) Varieties

Johnstone Omukhulu Neondo, Amos Emitati Alakonya, Jonathan Matheka, Joel Okoyo Masanga, Remmy Wekesa Kasili

Published in Journal of Plant Sciences (Volume 4, Issue 4)

Received: 5 July 2016 Accepted: 18 July 2016 Published: 3 August 2016

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Abstract

In vitro regeneration of open pollinated varieties (OPVs) Kakamega Striga Tolerant Population 94 (KSTP’94) and ‘Namba Nane’ alongside a tropical inbred line (CML144) was evaluated using immature zygotic embryos as explants. Four callus induction media (CIM) regimes; Murashige and Skoog (MS), Linsmaier and Skoog (LS), Chu (N6) and N6*(N6 medium fortified with 0.35 gL^-1 L-proline and 0.8 mgL^-1 AgNO3) were evaluated for their potential to induce callus in the three genotypes. All the media were supplemented with sucrose and five levels of 2, 4-Dichlorophenoxyacetic acid (2, 4-D) (0.5, 1.0, 1.5, 2.0 and 2.5 mgL^-1). Resulting calli were matured on MS and N6 basal media supplemented with 60 g/L sucrose and similar concentration levels (0.5, 1.0, 1.5, 2.0 and 2.5 mgL^-1) of 2, 4-D while the subsequent embryogenic calli were regenerated on hormone-free media. Transformability of these varieties was assessed via histochemical analysis of β-glucuronidase (GUS) reporter gene following Agrobacterium-mediated transformation. Statistical analyses were done using Statistical Analysis Software (SAS) and Graphpad Prism softwares with mean separations achieved at 95% confidence intervals. Of the 2 OPVs, KSTP’94 recorded the highest callus induction frequency (84.4%) while Namba Nane (45.6%) had the lowest. Similarly, KSTP, 94 had the highest mean of mature somatic embryos (59.7%) while Namba Nane recorded the lowest (16.4%). Assessment of regeneration frequencies from embryogenic calli revealed no significant differences among the 3 lines although CML 144 had the highest mean number of juvenile plantlets (36.7%). Analysis of transformation frequency (upon selection of calli on media with basta) showed that Namba Nane recorded the lowest transformation frequency (average 13.5%) some words missing. Transformation frequency (based on GUS positive calli) of these varieties ranged from 0.8 to 2.1%. This work therefore provides an empirical platform for potential introduction of useful genes into these varieties.

Published in	Journal of Plant Sciences (Volume 4, Issue 4)
DOI	10.11648/j.jps.20160404.14
Page(s)	80-87
Creative Commons	This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.
Copyright	Copyright © The Author(s), 2016. Published by Science Publishing Group

Keywords

Callus Induction, Embryogenesis, Plant Regeneration, Open Pollinated Varieties, GUS

References

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Johnstone Omukhulu Neondo, Amos Emitati Alakonya, Jonathan Matheka, Joel Okoyo Masanga, Remmy Wekesa Kasili. (2016). Transformation and Regeneration Protocol for Two Farmer Preferred Open Pollinated Tropical Maize (Zea Mays) Varieties. Journal of Plant Sciences, 4(4), 80-87. https://doi.org/10.11648/j.jps.20160404.14

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ACS Style

Johnstone Omukhulu Neondo; Amos Emitati Alakonya; Jonathan Matheka; Joel Okoyo Masanga; Remmy Wekesa Kasili. Transformation and Regeneration Protocol for Two Farmer Preferred Open Pollinated Tropical Maize (Zea Mays) Varieties. J. Plant Sci. 2016, 4(4), 80-87. doi: 10.11648/j.jps.20160404.14

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AMA Style

Johnstone Omukhulu Neondo, Amos Emitati Alakonya, Jonathan Matheka, Joel Okoyo Masanga, Remmy Wekesa Kasili. Transformation and Regeneration Protocol for Two Farmer Preferred Open Pollinated Tropical Maize (Zea Mays) Varieties. J Plant Sci. 2016;4(4):80-87. doi: 10.11648/j.jps.20160404.14

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@article{10.11648/j.jps.20160404.14,
  author = {Johnstone Omukhulu Neondo and Amos Emitati Alakonya and Jonathan Matheka and Joel Okoyo Masanga and Remmy Wekesa Kasili},
  title = {Transformation and Regeneration Protocol for Two Farmer Preferred Open Pollinated Tropical Maize (Zea Mays) Varieties},
  journal = {Journal of Plant Sciences},
  volume = {4},
  number = {4},
  pages = {80-87},
  doi = {10.11648/j.jps.20160404.14},
  url = {https://doi.org/10.11648/j.jps.20160404.14},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.jps.20160404.14},
  abstract = {In vitro regeneration of open pollinated varieties (OPVs) Kakamega Striga Tolerant Population 94 (KSTP’94) and ‘Namba Nane’ alongside a tropical inbred line (CML144) was evaluated using immature zygotic embryos as explants. Four callus induction media (CIM) regimes; Murashige and Skoog (MS), Linsmaier and Skoog (LS), Chu (N6) and N6*(N6 medium fortified with 0.35 gL-1 L-proline and 0.8 mgL-1 AgNO3) were evaluated for their potential to induce callus in the three genotypes. All the media were supplemented with sucrose and five levels of 2, 4-Dichlorophenoxyacetic acid (2, 4-D) (0.5, 1.0, 1.5, 2.0 and 2.5 mgL-1). Resulting calli were matured on MS and N6 basal media supplemented with 60 g/L sucrose and similar concentration levels (0.5, 1.0, 1.5, 2.0 and 2.5 mgL-1) of 2, 4-D while the subsequent embryogenic calli were regenerated on hormone-free media. Transformability of these varieties was assessed via histochemical analysis of β-glucuronidase (GUS) reporter gene following Agrobacterium-mediated transformation. Statistical analyses were done using Statistical Analysis Software (SAS) and Graphpad Prism softwares with mean separations achieved at 95% confidence intervals. Of the 2 OPVs, KSTP’94 recorded the highest callus induction frequency (84.4%) while Namba Nane (45.6%) had the lowest. Similarly, KSTP, 94 had the highest mean of mature somatic embryos (59.7%) while Namba Nane recorded the lowest (16.4%). Assessment of regeneration frequencies from embryogenic calli revealed no significant differences among the 3 lines although CML 144 had the highest mean number of juvenile plantlets (36.7%). Analysis of transformation frequency (upon selection of calli on media with basta) showed that Namba Nane recorded the lowest transformation frequency (average 13.5%) some words missing. Transformation frequency (based on GUS positive calli) of these varieties ranged from 0.8 to 2.1%. This work therefore provides an empirical platform for potential introduction of useful genes into these varieties.},
 year = {2016}
}

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TY  - JOUR
T1  - Transformation and Regeneration Protocol for Two Farmer Preferred Open Pollinated Tropical Maize (Zea Mays) Varieties
AU  - Johnstone Omukhulu Neondo
AU  - Amos Emitati Alakonya
AU  - Jonathan Matheka
AU  - Joel Okoyo Masanga
AU  - Remmy Wekesa Kasili
Y1  - 2016/08/03
PY  - 2016
N1  - https://doi.org/10.11648/j.jps.20160404.14
DO  - 10.11648/j.jps.20160404.14
T2  - Journal of Plant Sciences
JF  - Journal of Plant Sciences
JO  - Journal of Plant Sciences
SP  - 80
EP  - 87
PB  - Science Publishing Group
SN  - 2331-0731
UR  - https://doi.org/10.11648/j.jps.20160404.14
AB  - In vitro regeneration of open pollinated varieties (OPVs) Kakamega Striga Tolerant Population 94 (KSTP’94) and ‘Namba Nane’ alongside a tropical inbred line (CML144) was evaluated using immature zygotic embryos as explants. Four callus induction media (CIM) regimes; Murashige and Skoog (MS), Linsmaier and Skoog (LS), Chu (N6) and N6*(N6 medium fortified with 0.35 gL-1 L-proline and 0.8 mgL-1 AgNO3) were evaluated for their potential to induce callus in the three genotypes. All the media were supplemented with sucrose and five levels of 2, 4-Dichlorophenoxyacetic acid (2, 4-D) (0.5, 1.0, 1.5, 2.0 and 2.5 mgL-1). Resulting calli were matured on MS and N6 basal media supplemented with 60 g/L sucrose and similar concentration levels (0.5, 1.0, 1.5, 2.0 and 2.5 mgL-1) of 2, 4-D while the subsequent embryogenic calli were regenerated on hormone-free media. Transformability of these varieties was assessed via histochemical analysis of β-glucuronidase (GUS) reporter gene following Agrobacterium-mediated transformation. Statistical analyses were done using Statistical Analysis Software (SAS) and Graphpad Prism softwares with mean separations achieved at 95% confidence intervals. Of the 2 OPVs, KSTP’94 recorded the highest callus induction frequency (84.4%) while Namba Nane (45.6%) had the lowest. Similarly, KSTP, 94 had the highest mean of mature somatic embryos (59.7%) while Namba Nane recorded the lowest (16.4%). Assessment of regeneration frequencies from embryogenic calli revealed no significant differences among the 3 lines although CML 144 had the highest mean number of juvenile plantlets (36.7%). Analysis of transformation frequency (upon selection of calli on media with basta) showed that Namba Nane recorded the lowest transformation frequency (average 13.5%) some words missing. Transformation frequency (based on GUS positive calli) of these varieties ranged from 0.8 to 2.1%. This work therefore provides an empirical platform for potential introduction of useful genes into these varieties.
VL  - 4
IS  - 4
ER  -

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Author Information

Johnstone Omukhulu Neondo

Institute for Biotechnology Research, Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya
Amos Emitati Alakonya

Institute for Biotechnology Research, Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya
Jonathan Matheka

Biotechnology Unit, International Institute of Tropical Agriculture, Nairobi, Kenya
Joel Okoyo Masanga

Plant Transformation Laboratory, Department of Biochemistry and Biotechnology, Kenyatta University, Nairobi, Kenya
Remmy Wekesa Kasili

Institute for Biotechnology Research, Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya

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APA Style

Johnstone Omukhulu Neondo, Amos Emitati Alakonya, Jonathan Matheka, Joel Okoyo Masanga, Remmy Wekesa Kasili. (2016). Transformation and Regeneration Protocol for Two Farmer Preferred Open Pollinated Tropical Maize (Zea Mays) Varieties. Journal of Plant Sciences, 4(4), 80-87. https://doi.org/10.11648/j.jps.20160404.14

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ACS Style

Johnstone Omukhulu Neondo; Amos Emitati Alakonya; Jonathan Matheka; Joel Okoyo Masanga; Remmy Wekesa Kasili. Transformation and Regeneration Protocol for Two Farmer Preferred Open Pollinated Tropical Maize (Zea Mays) Varieties. J. Plant Sci. 2016, 4(4), 80-87. doi: 10.11648/j.jps.20160404.14

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AMA Style

Johnstone Omukhulu Neondo, Amos Emitati Alakonya, Jonathan Matheka, Joel Okoyo Masanga, Remmy Wekesa Kasili. Transformation and Regeneration Protocol for Two Farmer Preferred Open Pollinated Tropical Maize (Zea Mays) Varieties. J Plant Sci. 2016;4(4):80-87. doi: 10.11648/j.jps.20160404.14

Copy | Download

@article{10.11648/j.jps.20160404.14,
  author = {Johnstone Omukhulu Neondo and Amos Emitati Alakonya and Jonathan Matheka and Joel Okoyo Masanga and Remmy Wekesa Kasili},
  title = {Transformation and Regeneration Protocol for Two Farmer Preferred Open Pollinated Tropical Maize (Zea Mays) Varieties},
  journal = {Journal of Plant Sciences},
  volume = {4},
  number = {4},
  pages = {80-87},
  doi = {10.11648/j.jps.20160404.14},
  url = {https://doi.org/10.11648/j.jps.20160404.14},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.jps.20160404.14},
  abstract = {In vitro regeneration of open pollinated varieties (OPVs) Kakamega Striga Tolerant Population 94 (KSTP’94) and ‘Namba Nane’ alongside a tropical inbred line (CML144) was evaluated using immature zygotic embryos as explants. Four callus induction media (CIM) regimes; Murashige and Skoog (MS), Linsmaier and Skoog (LS), Chu (N6) and N6*(N6 medium fortified with 0.35 gL-1 L-proline and 0.8 mgL-1 AgNO3) were evaluated for their potential to induce callus in the three genotypes. All the media were supplemented with sucrose and five levels of 2, 4-Dichlorophenoxyacetic acid (2, 4-D) (0.5, 1.0, 1.5, 2.0 and 2.5 mgL-1). Resulting calli were matured on MS and N6 basal media supplemented with 60 g/L sucrose and similar concentration levels (0.5, 1.0, 1.5, 2.0 and 2.5 mgL-1) of 2, 4-D while the subsequent embryogenic calli were regenerated on hormone-free media. Transformability of these varieties was assessed via histochemical analysis of β-glucuronidase (GUS) reporter gene following Agrobacterium-mediated transformation. Statistical analyses were done using Statistical Analysis Software (SAS) and Graphpad Prism softwares with mean separations achieved at 95% confidence intervals. Of the 2 OPVs, KSTP’94 recorded the highest callus induction frequency (84.4%) while Namba Nane (45.6%) had the lowest. Similarly, KSTP, 94 had the highest mean of mature somatic embryos (59.7%) while Namba Nane recorded the lowest (16.4%). Assessment of regeneration frequencies from embryogenic calli revealed no significant differences among the 3 lines although CML 144 had the highest mean number of juvenile plantlets (36.7%). Analysis of transformation frequency (upon selection of calli on media with basta) showed that Namba Nane recorded the lowest transformation frequency (average 13.5%) some words missing. Transformation frequency (based on GUS positive calli) of these varieties ranged from 0.8 to 2.1%. This work therefore provides an empirical platform for potential introduction of useful genes into these varieties.},
 year = {2016}
}

Copy | Download

TY  - JOUR
T1  - Transformation and Regeneration Protocol for Two Farmer Preferred Open Pollinated Tropical Maize (Zea Mays) Varieties
AU  - Johnstone Omukhulu Neondo
AU  - Amos Emitati Alakonya
AU  - Jonathan Matheka
AU  - Joel Okoyo Masanga
AU  - Remmy Wekesa Kasili
Y1  - 2016/08/03
PY  - 2016
N1  - https://doi.org/10.11648/j.jps.20160404.14
DO  - 10.11648/j.jps.20160404.14
T2  - Journal of Plant Sciences
JF  - Journal of Plant Sciences
JO  - Journal of Plant Sciences
SP  - 80
EP  - 87
PB  - Science Publishing Group
SN  - 2331-0731
UR  - https://doi.org/10.11648/j.jps.20160404.14
AB  - In vitro regeneration of open pollinated varieties (OPVs) Kakamega Striga Tolerant Population 94 (KSTP’94) and ‘Namba Nane’ alongside a tropical inbred line (CML144) was evaluated using immature zygotic embryos as explants. Four callus induction media (CIM) regimes; Murashige and Skoog (MS), Linsmaier and Skoog (LS), Chu (N6) and N6*(N6 medium fortified with 0.35 gL-1 L-proline and 0.8 mgL-1 AgNO3) were evaluated for their potential to induce callus in the three genotypes. All the media were supplemented with sucrose and five levels of 2, 4-Dichlorophenoxyacetic acid (2, 4-D) (0.5, 1.0, 1.5, 2.0 and 2.5 mgL-1). Resulting calli were matured on MS and N6 basal media supplemented with 60 g/L sucrose and similar concentration levels (0.5, 1.0, 1.5, 2.0 and 2.5 mgL-1) of 2, 4-D while the subsequent embryogenic calli were regenerated on hormone-free media. Transformability of these varieties was assessed via histochemical analysis of β-glucuronidase (GUS) reporter gene following Agrobacterium-mediated transformation. Statistical analyses were done using Statistical Analysis Software (SAS) and Graphpad Prism softwares with mean separations achieved at 95% confidence intervals. Of the 2 OPVs, KSTP’94 recorded the highest callus induction frequency (84.4%) while Namba Nane (45.6%) had the lowest. Similarly, KSTP, 94 had the highest mean of mature somatic embryos (59.7%) while Namba Nane recorded the lowest (16.4%). Assessment of regeneration frequencies from embryogenic calli revealed no significant differences among the 3 lines although CML 144 had the highest mean number of juvenile plantlets (36.7%). Analysis of transformation frequency (upon selection of calli on media with basta) showed that Namba Nane recorded the lowest transformation frequency (average 13.5%) some words missing. Transformation frequency (based on GUS positive calli) of these varieties ranged from 0.8 to 2.1%. This work therefore provides an empirical platform for potential introduction of useful genes into these varieties.
VL  - 4
IS  - 4
ER  -

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