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Microparticle bombardment of Stylosanthes guianensis: transformation parameters and expression of a methionine-rich 2S albumin gene

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Abstract

An effective protocol to generate stable transformants of the tropical forage legume Stylosanthes guianensis (Aubl.) Sw. in a selection-free system was developed. Based on transient reporter gene expression, we have obtained transformation rates of 3.47% using 30-day-old calli as target, 1300 psi helium pressure, 12.5 cm microprojectile flight distance, 10–20 mm distance between macrocarrier membrane and stopping screen and 10–20 mm gap distance between the shock wave generator and the macrocarrier. These parameters were utilized to produce transgenic S. guianensis plants expressing Be2S1 from Bertholletia excelsa that codes for a methionine-rich storage protein driven by a green-tissue specific promoter, Ats1 from Arabidopsis thaliana. Transgenic plants were identified by a PCR-based high-throughput screen in a selective agent-free system, employing pools of 20–50 regenerating shoots. The integration of the exogenous gene in the host genome was confirmed by Southern blot analysis of PCR-positive plants. The expression of the introduced gene was confirmed in leaf tissue of transgenic plants by Northern and Western blot analyses. Immunoblots of cellular fractions showed that BE2S1 expressed in Stylosanthes is mainly targeted to the vacuoles.

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Abbreviations

AMV:

Alfalfa mosaic virus

BAP:

6-Benzylaminopurine

BCIP:

5-Bromo-4-chloro-3-indolyl-phosphate

CAMV:

Cauliflower mosaic virus

ELISA:

Enzyme-linked immunosorbent assay

GUS:

β-Glucuronidase

NBT:

Nitro blue tetrazolium

NEO:

Neomycin phosphotransferase

NOS:

Nopaline synthase

Rubisco:

Ribulose 1,5-bisphosphate carboxylase/oxygenase

SDS-PAGE:

Sodium dodecylsulphate-polyacrylamide gel electrophoresis

X-gluc:

5-Bromo-4-chloro-3-indolyl glucuronide

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Acknowledgements

This research was funded by FAPESP 98/11270-7 grant to MLCV and by CNPq 141294/97-3 graduate fellowship grant to V.M.Q. We thank Prof. Dr. Flávio C. A. Tavares, Dr. Luiz Humberto Gomes and Dr. Keila M. R. Duarte for the assistance with antibody generation.

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  1. V. M. Quecini

    Present address: Centro de Pesquisa e Desenvolvimento de Recursos Genéticos, Instituto Agronômico, P.O. Box 28, CEP13001-970, Campinas, SP, Brazil

Authors and Affiliations

  1. Departamento de Genética, ESALQ, Universidade de São Paulo, P.O. Box 83, CEP 13400-970, Piracicaba, SP, Brazil

    V. M. Quecini, C. A. Oliveira & M. L. C. Vieira

  2. Laboratório Nacional de Luz Síncrotron, Caixa Postal 6192, CEP 13084-971, Campinas, SP, Brazil

    A. C. Alves

  3. Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica, Final Av. W3 Norte, 70770-900, Brasília, DF, Brazil

    F. J. L. Aragão, E. L. Rech, E. R. P. Almeida & E. S. Gander

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  1. V. M. Quecini
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Quecini, V.M., Alves, A.C., Oliveira, C.A. et al. Microparticle bombardment of Stylosanthes guianensis: transformation parameters and expression of a methionine-rich 2S albumin gene. Plant Cell Tiss Organ Cult 87, 167–179 (2006). https://doi.org/10.1007/s11240-006-9151-x

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