full issue - Association of Biotechnology and Pharmacy
full issue - Association of Biotechnology and Pharmacy
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Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong>Vol. 5 (2) 1149-1156 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)may over come two limitations <strong>of</strong> currenttransgenic technology i.e., unpredictabletransgene expression <strong>and</strong> effect <strong>of</strong> the mutationinduced by transgene integration. Selection <strong>of</strong>multiple copy gene as targeted site for HR basedintegration may result in multicopy transgenics.However, this is a rare possibility, when chances<strong>of</strong> getting one HR event is in an order <strong>of</strong> 10 -4chances <strong>of</strong> getting two integration events in asingle cell will be in an order <strong>of</strong> 10 -8 .Identification <strong>of</strong> gene targeted eventsLow frequency <strong>of</strong> homologousrecombination in comparison to r<strong>and</strong>om T-DNAintegration is a major limitation to find a rare genetargetedtransgenic event. Earlier Terada et al.,has used a positive-negative selection foridentification <strong>of</strong> six gene targeting events in rice(30). However, use <strong>of</strong> negative selection such asdiphtheria toxin (DT) has a disadvantage becausethe presence <strong>of</strong> even single toxin moleculeproduced by transient expression is sufficient tokill a cell. Losing any cell having gene integrationby HR, because <strong>of</strong> transiently expressed DT willbe counterproductive when GT frequency is inan order <strong>of</strong> 10 -4 . To overcome this problem anegative screening strategy in combination with1153a positive selection marker nptII was employedin the present study. The T-DNA carries GUSgene expression cassette, which is placed out <strong>of</strong>F3F gene sequence used for homologousrecombination (Fig. 1). Thus the r<strong>and</strong>omintegration transgenic events will have a functionalGUS expression cassette while transgenicdeveloped by homologous recombination will bedevoid <strong>of</strong> GUS gene (Fig. 1, 3). R<strong>and</strong>omintegration events were screened out by GUSassay at an early stage <strong>of</strong> plant regeneration.However, with this approach non-transgenic <strong>and</strong>partial T-DNA integration (without GUS) eventsalso show GUS-negative results. Hence, putativetransgenic plants need further molecular analysis.This limitation was overcome by employing a PCRscreening method earlier developed in ourlaboratory (29) that overcomes the limitation <strong>of</strong>false positive amplification arising fromcontaminating Agrobacterium. Combination <strong>of</strong>negative screening with modified PCR protocolgreatly simplified the identification <strong>of</strong> rare HRevents. Use <strong>of</strong> negative screening methodologymay prove useful in plants that have long callusphase during regeneration. R<strong>and</strong>om integrationevents can be screened out during early callusphase.Fig. 3 GUS assay <strong>of</strong> leaf from putative transgenic brinjal plants. A. Transgenic plants developed throughr<strong>and</strong>om T-DNA integration (GUS positive), B. Transgenic plants developed through targeted gene integration(GUS negative).Bt gene targeting in Brinjal