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Molecular Plant Breeding Provisional Publishing
Molecular Plant Breeding 2012, Vol.3, No.5, 50
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56
http://mpb.sophiapublisher.com
50
Research Report Open Access
Optimization of Minimal Inhibitory Dose of Selective Agent (Basta) for
Selection of Transgenics in Sugarcane
Siddra Ijaz , Naweed Anjum , Iqrar Ahmad Rana , Iqrar Ahmad Khan
Centre of Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture Faisalabad, Pakistan
Corresponding authors email:
siddraijazkhan @ yahoo.com;
Authors
Molecular Plant Breeding, 2012, Vol.3, No.5 doi: 10.5376/mpb.2012.03.0005
Received: 13 Mar., 2012
Accepted: 16 Apr., 2012
Published: 30 Apr., 2012
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work is properly cited.
Preferred citation for this article:
Ijaz et al., 2012, Optimization of Minimal Inhibitory Dose of Selective Agent (Basta) for Selection of Transgenics in Sugarcane, Molecular Plant Breeding,
Vol.3, No.5 50
-
56 (doi: 10.5376/mpb.2012.03.0005)
Abstract
Success rate in the selection of putative transformants is mainly dependent upon the dose rate of selective agent. Selection
pressure determines the survival frequency of transformant and non transformant cells. Dose of selective agent, which is lethal for the
non transformants, would be the optimum for the transgenic selection. We investigated the optimum dose of basta (selective agent),
in two sugarcane genotypes viz., S-2003-us-359 and S-2003-us-127, in which we have already established a proficient
in vitro
regeneration system (Ijaz et al., 2012; Anjum et al., 2012). Different dosages of basta viz., 1 mg/L, 3 mg/L, 5 mg/L, 7 mg/L and 10
mg/L were studied for regeneration media (RM) of both genotypes. For determining the optimal inhibitory dose of basta on
regeneration, 21 days old calli of S-2003-us-359 were induced on CIM1, 28 days old calli of S-2003-us-127 were induced on CIM2
and were shifted to RSM1 and RSM2 respectively. In genotype S-2003-us-359, early callus death was observed on RSM having 10
mg/L basta, and RSM having 5 mg/L, 7 mg/L, 10 mg/L basta proved to be lethal for S-2003-us-127 with early death of calli. For
genotype S-2003-us-127, among 5 mg/L, 7 mg/L and 10 mg/L basta levels, 5 mg/L basta was selected for regeneration media as
optimal dose for the selection of transgenics of this genotype. For transgenic selection in both sugarcane genotypes, the affectivity of
selected optimal doses of basta was checked. Twenty one (21) days old calli of S-2003-us-359 and 28 days old calli of S-2003-us-127
were bombarded with
bar
gene and plant were selected at 10 mg/L and 5 mg/L of basta respectively. Putative transgenic plants with
integrated copy of
bar
gene were confirmed with PCR analysis.
Keywords
Basta; Bar gene; Sugarcane; Transgenics
Background
Transformation of crop genomes is used for many
purposes such as increased nutritive value, plant
disease resistance, insect resistance, herbicide
resistance, and for high yield. The success of the
genetic transformation process is monitored through
the following 3 steps: the proof for DNA integration,
protein expression and transmission of the transgene
into its progenies. Practically, during genetic
transformation, foreign gene is transferred into target
tissues, which contains thousands of cells, and only a
few cells will become transgenic or will have the
transgene stably integrated into its genome. It is very
important to isolate these transformed cells from the
majority of untransformed cells by using a selection
agent. The transform cells should carry a selectable
marker gene, which will make the cell survive on a
particular selection agent. It was reported that there
are approximately 50 selectable marker genes being
used or being developed in transgenic plant research
(Miki and McHugh, 2004). In genetic transformation
study the
bar
gene has been widely used as an effective
selectable marker in many crop species (Gordon-Kamm
et al., 1990; Akama et al., 1995).
Bar
gene was isolated
from Streptomyces hygroscopicus (Block et al., 1987)
and confers resistance to phosphinothricin, the active
ingredient for the herbicides bialaphos, Liberty and
Basta, an analogue of glutamate, a competitive
inhibitor of the enzyme glutamine synthetase.
Phosphinothricin acetyltransferase, coded by
bar
gene
inactivates phosphinothricin by acetylation.
The concentration of selection agents need to be
carefully chosen to avoid either being too low and
thereby allowing undesirable numbers of ‘escapes’
to develop, or too high so that transformants expressing