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Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 116, Issue 2, pp 175–185 | Cite as

Using flow cytometry and cytological analyses to assess the genetic stability of somatic embryo-derived plantlets from embryogenic Musa acuminata Colla (AA) ssp. malaccensis cell suspension cultures

  • Rosa M. Escobedo-GraciaMedranoEmail author
  • Josefina I. Maldonado-Borges
  • Martha J. Burgos-Tan
  • Nina Valadez-González
  • José R. Ku-Cauich
Original Paper

Abstract

Flow cytometry and chromosome counts were used to analyze the genetic stability of plants regenerated via Musa acuminata Colla (AA) ssp. malaccensis embryogenic cell suspension (ECS) cultures. These cultures were initiated from immature zygotic embryos (IZE) on Murashige and Skoog medium using nine different plant growth regulator (PGR) treatments. Highest percentage of embryogenic calli (EC) formation occurred on media with 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D, 97 %), and 8.2 μM of picloram (Pi, 80 %) followed by 2.2 μM 2,4-D (75 %). Embryonic development was synchronized in liquid medium by filtration, and somatic embryo development was achieved with ECS aliquots overlaid on PGR-free medium. The EC medium composition and elapsed time of both short-term (~5 months old) and long-term (~2 years old) ECS cultures influenced plant regeneration, resulting in 65–99 % embryo germination and 50 to 100 % plant conversion. The mean 2C DNA content (1.23 ± 0.002 pg) and chromosome number (2n = 2x = 22) of M. acuminata ssp. malaccensis IZEs, seedlings and sucker plantlets were similar to the reported values. No significant differences were detected among IZEs before culturing, and none were found among the IZEs and leaves of control plants and the plants regenerated from short-term ECS lines when initiated with 2,4-D or Pi vis-à-vis the accession from which they originated. However, plants regenerated from the long-term ECS-L3 culture remained diploid, had the highest DNA content (2C = 1.283 ± 0.01 pg) and were clearly separate from the other regenerated and control plants.

Keywords

Flow cytometry Diploid banana Immature zygotic embryos Somaclonal variation Somatic embryogenesis 

Abbreviations

2,4-D

2,4-Dichlorophenoxyacetic acid

FCM

Flow cytometry

FACS

Fluorescence-activated cell sorter

IZE

Immature zygotic embryos

Pi

Picloram (4-amino-3, 5,6-trichloropicolinic acid)

PI

Propidium iodide

SE

Somatic embryogenesis

SEM

Scanning electron microscopy

SV

Somaclonal variation

Z

Zeatin (6-(4-hydroxy-3-methylbut-2-enylamino) purine)

Notes

Acknowledgments

The authors thank the following individuals: Dr. Jaroslav Dolezel at the Institute of Experimental Botany, Olomouc, Czech Republic for providing the Glycine max cv. Polanka seeds; Ir. I. Van den Houwe, curator of the Musa International Transit Centre at Katholieke Universiteit, Leuven, Belgium, for the plant material; INIFAP-CIR-SURESTE for the conservation field facilities for the in situ Musa collection as launched by the CICY at the INIFAP-Uxmal-Experimental Station-Yucatán, México; Dr. Goreti Campos and MSc. Angela Ku for their help with the scanning electron microscopy; IQA. Wilma A. González for the maintenance of ECS lines; and Ing. Fernando Contreras for assisting with the greenhouse work. This work was funded by SAGARPA-CONACYT Applied Research Project # 0048160 and by CONACYT Basic Science Research Project # 0060838 (to the corresponding author), and studentship # 169880 as awarded to the second author, and a studentship supported by the SAGARPA-CONACYT project (# 0048160) to the third author.

Supplementary material

11240_2013_394_MOESM1_ESM.pdf (146 kb)
Supplementary material 1 (PDF 147 kb)
11240_2013_394_MOESM2_ESM.pdf (344 kb)
Supplementary material 2 (PDF 345 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Rosa M. Escobedo-GraciaMedrano
    • 1
    Email author
  • Josefina I. Maldonado-Borges
    • 1
  • Martha J. Burgos-Tan
    • 1
  • Nina Valadez-González
    • 2
  • José R. Ku-Cauich
    • 1
  1. 1.Unidad de Bioquímica y Biología Molecular de PlantasCentro de Investigación Científica de Yucatán, A.C. MéridaMéridaMexico
  2. 2.Laboratorio de Hematología, Centro de Investigaciones Regionales. Dr. Hideyo NoguchiUniversidad Autónoma de YucatánMéridaMexico

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