Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 122, Issue 3, pp 605–616 | Cite as

A method to identify early-stage transgenic Medicago truncatula with improved physiological response to water deficit

  • A. Alcântara
  • R. S. Morgado
  • S. Silvestre
  • J. Marques da Silva
  • A. Bernardes da Silva
  • P. Fevereiro
  • S. S. AraújoEmail author
Original Paper


Phenotypic screening after transformation experiments aiming to identify lines with the enhanced/desired trait is still a time consuming process for most agricultural crops, especially when dealing with complex physiological responses such as water deficit. In this study we evaluated the suitability of non-destructive leaf gas-exchange analysis and imaging-PAM chlorophyll a fluorescence to select transgenic lines of Medicago truncatula expressing the Trehalose-6-Phosphate Synthase 1 (AtTPS1) from Arabidopsis thaliana with altered response to water deficit (WD) and WD recovery (WDR) in the early stages of the transformation process (T0). Primary transformants (T0) with different expression levels of a constitutive AtTPS1 construct were used. Additionally, we evaluated if the expression of the transgene could be correlated with the phenotype assessed. Among tested techniques and parameters measured, the net carbon assimilation (A) from gas-exchange analysis was the best parameter to early detect lines with WD and WDR improved performance, at the earliest stages of the transformation process. With this multidisciplinary approach, we selected 3 transgenic lines TPS7, TPS10 and TPS16 for further studies, which have higher or intermediate expression levels of the transgene and improved response to WD and WDR. This work will contribute to speed-up the identification of elite lines with confidence within a large number of individuals, thus reducing time, cost and labor associated with this plant improvement strategy.


Early-stage selection Legumes Trehalose metabolism Physiological responses T0 transformants Water deficit 



Net photosynthesis rate

Chl a

Chlorophyll a

Chl b

Chlorophyll b


Moderate water deficit


Photosynthetic active radiation


Reverse transcription quantitative PCR


Relative water content


Soil water content


Severe water deficit


Water deficit


Water deficit recovery


Well watered


Primary transformants




Effective quantum yield of the photosystem II


Leaf water potential



The financial support from Fundação para a Ciência e a Tecnologia (Lisbon, Portugal) is acknowledged through research projects PTDC/AGR-GPL/099866/2008, PTDC/AGR-GPL/110224/2009 and research unit GREEN-it “Bioresources for Sustainability” (UID/Multi/04551/2013). SSA acknowledges a grant by the CARIPLO Foundation (Milan, Italy), in scope of the Integrated Project ‘Advanced Priming Technologies for the Lombardy Agro-Seed Industry-PRIMTECH’ (Action 3, Code 2013-1727). The authors would like to thank to Prof. André Almeida (Ross University, St. Kitts and Nevis) for kindly reviewing the English language of this manuscript.

Supplementary material

11240_2015_793_MOESM1_ESM.ppt (143 kb)
Supplementary material 1 Figure S.1 – Representative PCR for the presence of the AtTPS1 fragment in regenerated transgenic M. truncatula lines. A 918 bp fragment was amplified in tested lines (TPS4, TPS7, TPS10, TPS14 and TPS 16) and positive control (C+, plasmid pBIN-2x35S-AtTPS1-t35S). No amplification was detected in non-transformed control (M910a) and non-template control (C-). M stands for molecular weight marker (1 kb DNA ladder) (PPT 141 kb)
11240_2015_793_MOESM2_ESM.doc (40 kb)
Supplementary material 2 (DOC 39 kb)


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Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  1. 1.Biosystems and Integrative Sciences Institute (BioISI)LisbonPortugal
  2. 2.Departamento de Biologia Vegetal, Faculdade de CiênciasUniversidade de LisboaLisbonPortugal
  3. 3.Plant Cell Biotechnology Laboratory, Instituto de Tecnologia Química e Biológica António Xavier (ITQB)Universidade Nova de Lisboa (UNL)OeirasPortugal
  4. 4.Unidade de Veterinária e Zootecnia, Instituto de Investigação Científica e Tropical (BIOTROP-CVZ)Avenida da Universidade TécnicaLisbonPortugal
  5. 5.Plant Biotechnology Laboratory, Department of Biology and Biotechnology ‘L. Spallanzani’Università degli Studi di PaviaPaviaItaly

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