GENETIC TRANSFORMATION STUDIES IN LETTUCE (Lactuca sativaL.) WITH npt-IIAND gusGENES
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Date
2014
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UHF,NAUNI
Abstract
ABSTRACT
Genetic transformation studies were carried out to standardize a protocol using
Agrobacterium-mediated gene transfer technique. High efficiency plant regeneration protocol is a
prerequisite of genetic transformation, hence the plant regeneration protocol was also standardized.
Two types of explants viz leaf and petiole were used for plant regeneration studies. High percentage
shoot regeneration in petiole (70.73%) and leaf (70.10%) explants were obtained on MS medium
containing 0.25mg/l BAP+0.10mg/l NAA and MS medium containing 1.00 mg/l BAP+0.10mg/l NAA
respectively. MS medium supplemented with 0.50mg/l IBA was found to be best for root regeneration
(68.00%). Kanamycin sensitivity experiment was carried out to study the effect of antibiotic on
relative growth of leaf and petiole explant and to select transgenic shoots during genetic
transformation experiment. Kanamycin concentration above 30mg/l was toxic to the explants on
selective shoot regeneration medium. Disarmed Agrobacterium tumefaciens LBA 4404 strain
containing a reporter β- glucuronidase [uidA (gus)] gene in binary vector (pBI 121) system along with
kanamycin resistance gene (npt-II) for selection in both bacteria and plant was used for genetic
transformation studies in lettuce. After co-cultivation only the transformed cells/tissues were able to
grow on selective shoot regeneration medium (25mg/lKanamycin and 400mg/l cefotaxime) whereas
non-transformed cells/tissues turned brown and died on the selective medium. Transformation
experiment could be scored as early as 5-6 weeks after selection. The effect of pre-culturing and cocultivation was studied on transformation frequency. Explants pre-cultured for 48 hours and cocultivated for 48 hours resulted in shoot regeneration. Putative transformed shoots obtained from leaf
and petiole explants, were able to grow on the selective medium containing 25mg/l Kanamycin. The
presence of npt-IIand uid A (gus)was confirmed by PCR using designed primers in thetransgenic
shoots. Out of 5 putative transformed shoots, 3 have shown the amplification of npt-IIand 4 shown the
amplification of uid A (gus)genes. Expression of uid A (gus) gene was studied in the PCR positive
shoots by β-glucuronidase assay using biochemical and histochemical methods. The transformed
shoots were GUS positive. A protocol for genetic transformation in lettuce with npt-IIand gusgenes
has been standardized.
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