full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and PharmacyVol. 5 (2) 1149-1156 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)may over come two limitations of currenttransgenic technology i.e., unpredictabletransgene expression and effect of the mutationinduced by transgene integration. Selection ofmultiple copy gene as targeted site for HR basedintegration may result in multicopy transgenics.However, this is a rare possibility, when chancesof getting one HR event is in an order of 10 -4chances of getting two integration events in asingle cell will be in an order of 10 -8 .Identification of gene targeted eventsLow frequency of homologousrecombination in comparison to random T-DNAintegration is a major limitation to find a rare genetargetedtransgenic event. Earlier Terada et al.,has used a positive-negative selection foridentification of six gene targeting events in rice(30). However, use of negative selection such asdiphtheria toxin (DT) has a disadvantage becausethe presence of even single toxin moleculeproduced by transient expression is sufficient tokill a cell. Losing any cell having gene integrationby HR, because of transiently expressed DT willbe counterproductive when GT frequency is inan order of 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 ofF3F gene sequence used for homologousrecombination (Fig. 1). Thus the randomintegration transgenic events will have a functionalGUS expression cassette while transgenicdeveloped by homologous recombination will bedevoid of GUS gene (Fig. 1, 3). Randomintegration events were screened out by GUSassay at an early stage of plant regeneration.However, with this approach non-transgenic andpartial 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 offalse positive amplification arising fromcontaminating Agrobacterium. Combination ofnegative screening with modified PCR protocolgreatly simplified the identification of rare HRevents. Use of negative screening methodologymay prove useful in plants that have long callusphase during regeneration. Random integrationevents can be screened out during early callusphase.Fig. 3 GUS assay of leaf from putative transgenic brinjal plants. A. Transgenic plants developed throughrandom T-DNA integration (GUS positive), B. Transgenic plants developed through targeted gene integration(GUS negative).Bt gene targeting in Brinjal