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Development of Marker Free Transgenic (MFT) Crops

Sudhir Kumar1 & Mahesh Rao2
1. Scientist, ICAR RC for NEH Region Manipur Centre, Imphal-795004
2. Scientist, NRC Plant Biotechnology, New Delhi- 110012

Recent advance in biotechnology has offer a varieties of tools like, protoplast culture, anther culture,
molecular markers for use in crop improvement for human kind. The discovery of Recombinant
DNA technology has further open the scope for improvement of economic important traits in different
crops plant. A crop plant which has been artificially inserted with a gene or genes instead of acquiring
through pollination is referred as transgenic plant or often termed as genetically modified or GM
crops. Creation of transgenic plant requires creation of suitable gene constructs and transformation of
this gene construct into plant cells (genetic transformation). Transformation event could be performed
by either direct methods like biolistic or through agrobacterium mediated vector. Selection of
transformed calli could be achieved by using a selectable antibiotic marker system and regeneration of
fertile plant from positive tansformatnt plant. Antibiotic resistance markers or herbicide resistance
markers (eg. bar) are being routinely used for selection of tranformant cell and tissues. Once the
tansformant is selected and regenerated the maintenance of markers in transgenic plant is not
necessary since it could lead to development of super pathogenic races or super weed which is a
serious concern over the bio-safety issue. Development of marker free transgenic plant has also
several advantage as successive round of transformation useful transgenic can be stacked, activating
signals for transcriptional silencing. (Hohn et al., 2001). There are several strategies to exclude the
selection gene for marker-free plants in transgenic generations, such as co-transformation, sitespecific recombination multi-autotransformation vector transposition system (Goldsbrough et al.
1993) and homologous recombination (Puchta et al. 2000 ). among which co-transformation has been
widely used. The first successful example of maker free transgenic involved the P1 bacteriophage
recombinase (Cre) and its recognition site (lox) (Dale and OW 1991). In this pioneer work, a loxflanked hpt gene was removed from transgenic plants upon re-transformation with a construct

expressing the Cre-recombinase gene. The maize tansposon, Ac/Ds system has also being used for
selectable marker removal in tomato by using T-DNA with Ds-gus-Ds/Ac/nptII as tansformant.
During transposition events, T0 plant was place with gus and nptII genes in unlinked loci and further
segregation in T1 plant maker free transgenic were obtained (Goldsbrough, et al. 1993). Cotransformation could be performed by three different methods, first involve use of two different
vector carrying by two different agrobacterium strains, second method involve use of two vector in
same agrobacterium starain and third method use of two T-DNA within the same binary vector.
(Tuteja et al., 2012). Co- transformation with two independent T-DNA, one with selectable maker
gene in one construct and gene of interest in other T-DNA construct, and in further generation (T1)
marker free transgenic plant (with gene of interest but lacking marker gene) will be obtained due to
segregation . Alternative strategies for making free transgenic plant, breeders are using positive
selection markers that are consider to be environmentally safe. In this method tansformant cells has
certain metabolic advantage than non-tranformant cells in proliferation and growth. Several example
of positive selection has been reported in different crops such as, xylose isomerise gene (xylA) use in
potato and tomato and E.coli phosphomannose isomerise gene (pmi) has been widely used in maize,
rice , cassava and beet . In future there is need to develop a clean techniques for production of
marker free transgenic production.

References:
Dale, EC and David WO 1991 Gene transfer with subsequent removal of the selection gene from the
host genome. Proc. Natl. Acad. Sci. USA 88 10558–10562.
Dale, E.C. and D.W. OW. ( 1991) Gene transfer with subsequent removal of the selection gene from
the host genome. Proc. Natl. Acad. Sci. USA 88 :10558–10562.
Goldsbrough, A.P., Lastrella, C.N. and Yoder, J.I.( 1993) Transposition-mediated re-positioning and
subsequent elimination of marker genes from transgenic tomatoes. Biotechnology 11: 1286–1292

Hohn, B., Levy, A.A. and Puchta, H. (2001) Elimination of selection markers from transgenic plants.
Curr. Opin. Biotechnol.12 139–143.
Puchta, H. (2000). Removing selectable marker genes: taking the shortcut. Trend. Plant Sci. 5: 273–
274.
Tuteja, N., Verma, S., Sahoo, R.K., Raveender, S. and Reddy, I.B.L. (2012). Recent advances in
development of marker free-transgenic pant: Regulation and biosafety concern. J. Biosci. 37(1): 167–
197.