ORIGINAL PAPER
Induced ectopic expression of At-CBF1 in marker-free transgenic
tomatoes confers enhanced chilling tolerance
Shweta Singh
•
Meenal Rathore
•
Danswrang Goyary
•
Rupesh Kumar Singh
•
Sivalingam Anandhan
•
Dinesh K. Sharma
•
Zakwan Ahmed
Received: 19 November 2010 / Revised: 6 January 2011 / Accepted: 10 January 2011 / Published online: 3 February 2011
Ó Springer-Verlag 2011
Abstract In an attempt to improve chilling stress toler-
ance, an Arabidopsis C-repeat binding factor 1 (At-CBF1)
gene driven by the inducible promoter RD29A was co-
transferred into tomato var. Shalimar. Marker (NPTII)-free
transgenic were obtained in T
1
generation because of
unlinked integration of CBF1 and NPTII genes. Reverse
transcription-polymerase chain reaction confirmed the
expression of CBF1 in T
1
transgenic lines. Study of
expression pattern in T
1
transgenic line showed a gradual
increase with increasing chilling stress period and also
confirmed the reversibility of expression on removal of
stress. The transgenic plants exhibited no morphological
and agronomical differences as compared to non-trans-
formed plants. When young transgenic plants were exposed
to chilling stress (4°C) for 3 days, increased survival (50%)
was observed in transgenic lines than non-transformed
plants (10%). Transgenic plants subjected to the chilling
stress showed a significant decrease in membrane injury
index and lipid peroxidation and also increased signifi-
cantly free proline content in the leaf tissues as compared
to non-transformed plants. Thus, these findings indicate
that marker-free transgenic tomato plants expressing Ara-
bidopsis CBF1 gene provided protection and conferred
cold tolerance to transgenic tomato without any phenotypic
variation.
Keywords Agrobacterium tumifaciens Á Chilling
tolerance Á C-repeat binding factor-1 Á Transgenic tomato Á
Shalimar Á Co-transformation
Abbreviations
BAP 6-Benzylaminopurine
CBF1 C-repeat binding factor-1
CTAB Cetyl tri-methyl ammonium bromide
IAA Indole acetic acid
MII Membrane injury index
MS Murashige and Skoog
NPTII Neomycin phosphotransferase-II
RT-PCR Reverse transcription-polymerase chain
reaction
Introduction
Abiotic stresses, such as cold, drought, salinity, wind
velocity, humidity and heat, are the major environmental
factors that adversely affect the growth of plants, produc-
tivity and quality of crops (Djilianov et al. 2005). Abiotic
stress is the primary cause of crop loss worldwide, reducing
crop productivity for most major crop plants and thus
threatening the sustainability of agricultural industry. Cold,
drought and salinity stress are interconnected and affect the
water relations of a plant on the cellular as well as whole
plant level (Beck et al. 2007), leading to a series of mor-
phological, physiological and biochemical changes (Baker
et al. 1994). Among various abiotic stresses, low temper-
ature is the major constraint effecting plants by dehydration
Communicated by A. Feher.
S. Singh Á M. Rathore Á D. Goyary (&) Á
R. K. Singh Á S. Anandhan Á Z. Ahmed
Molecular Biology and Genetic Engineering Division,
Defence Institute of Bio-Energy Research, Haldwani 263 139,
Uttarakhand, India
e-mail: dgoyary@gmail.com
D. K. Sharma
Department of Zoology, HNB Garhwal University,
SRT Campus, Badshahithal, Tehri, Uttarakhand, India
123
Plant Cell Rep (2011) 30:1019–1028
DOI 10.1007/s00299-011-1007-0