African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12392

Full Length Research Paper

Isolation and regeneration of protoplasts from leaf explants of Rhyncholaelia digbyana

Luis Alberto Mota-Narváez
  • Luis Alberto Mota-Narváez
  • Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, A.C., Sede Sureste, km 5.5 Sierra Papacal-Chuburna Puerto, Parque Científico y Tecnológico de Yucatán, Postal code 97302 Mérida, Yucatán, México.
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Gilbert José Herrera-Cool
  • Gilbert José Herrera-Cool
  • Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, A.C., Sede Sureste, km 5.5 Sierra Papacal-Chuburna Puerto, Parque Científico y Tecnológico de Yucatán, Postal code 97302 Mérida, Yucatán, México.
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Teresa del Rosario Ayora-Talavera
  • Teresa del Rosario Ayora-Talavera
  • Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, A.C., Sede Sureste, km 5.5 Sierra Papacal-Chuburna Puerto, Parque Científico y Tecnológico de Yucatán, Postal code 97302 Mérida, Yucatán, México.
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Nancy Santana-Buzzy
  • Nancy Santana-Buzzy
  • Centro de Investigación Científica de Yucatán, A.C., Unidad de bioquímica Calle 43 No. 130 x 32 y 34 Chuburná de Hidalgo, Postal code. 97205, Mérida, Yucatán, México.
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Guadalupe López-Puc
  • Guadalupe López-Puc
  • Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, A.C., Sede Sureste, km 5.5 Sierra Papacal-Chuburna Puerto, Parque Científico y Tecnológico de Yucatán, Postal code 97302 Mérida, Yucatán, México.
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  •  Received: 05 June 2018
  •  Accepted: 30 July 2018
  •  Published: 29 August 2018

Abstract

A protocol for the isolation and regeneration of protoplasts from leaf explant of Rhyncholaelia digbyana is presented. The protoplasts were isolated using hemicellulase enzymes at 1.5, 2.25, and 3% (w/v), pectinase at 0.5 and 0.75% (w/v) and cellulase at 1 and 2% (w/v). Protoplast counting was carried out with a Neubauer camera and an optical microscope at 40X, and viability was determined with Evans blue dye at 0.025% (w/v). The protoplasts were cultivated following the standard plate method, using the K&M media with 0.06 M of saccharose, 2.3% of Gelrite and plant growth regulators. It produced a yield of 386250±1875 protoplasts/g of tissue using an enzymatic combination of 1.5% (w/v) of hemicellulase, 0.5% (w/v) of pectinase and 1% (w/v) of cellulase with an incubation time of 4 h. The colonies were observed after two months of culture and the highest number of colonies (1.66±0.50) was obtained when the protoplasts were cultured in Kao medium with 4.53 µM of 2,4-Dichlorophenoxyacetic acid (2,4-D), 0.912 µM of Zeatin and 3.10 µM of 6-Benzylaminopurine (BA). The nuclear DNA content estimated by flow cytometry for R. digbyana was 27.39±3.8 pg of DNA equivalent to 26.79×109 pb and the number of mitotic chromosomes counted was 2n=40.

 

Key words: Isolation, in vitro culture, plant regeneration, flow cytometry.