Aspectos sobre el servicio

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4. On the following day, remove the assay solution and decolor the tissue in 70–100 % ethanol.

5. Observe under the light microscope and photograph (Fig. 2a–l ).

Fig. 2 Transient and stable GUS activities in various tissues of transgenic plants. Transient GUS assay in ( a ) non-transformed (control) explants not showing GUS activity and ( b) cotyledon explants showing GUS activ-ity after 2 days of cocultivation with Agrobacterium tumefaciens EHA105 (pCAMBIA2301). Stable GUS assay in roots from non-transformed ( c ) and transformed ( d ) plants; shoots from non-transformed ( e ) and transformed ( f ) plant; anthers from non-transformed ( g ) and transformed ( h ) plant; pollen grains from non-transformed ( i ) and transformed ( j ) plant; and germinating seeds from non-transformed ( k ) and transformed ( l ) plant.

Transgenic plants were recovered and rooted on kanamycin-containing medium and germinated T ₁ seeds (Reproduced from Yadav et al. 2010 [ 16 ], with permission from Springer)

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4 Notes

1. Inoculation and cocultivation steps are crucial for the success of any transformation experiment. Bacterial concentration at 10 ⁶ cells/mL, bacterial inoculation time for 20 min, and cocultivation for 2 days in the presence of BAP 25.0 μM and acetosyringone 20.0 μM at pH 5.5 under light conditions were effective in improving transformation effi ciency.

2. Inclusion of a combination of thiol compounds, L -cysteine (400 mg/L), and DTT (1.0 mM) in the cocultivation medium completely inhibited the browning and necrosis of explants. It increased the percentage of explants showing intense transient GUS activity and also improved the survival of explants on the selection medium.

3. Kanamycin at a concentration of 5 mg/L completely inhibited rooting in non-transformed control plants indicating that root induction is more sensitive to kanamycin than shoot organo-genesis which is inhibited at 25 mg/L.

4. At the onset of pod maturation, the pods should be covered with polythene bags to prevent seed loss due to pod shattering.

5. GUS assay solution should be stored at −20 °C in small ali-quots of 1 mL to avoid repeated freezing and thawing. Before use, thaw at room temperature since the components degrade in heat immediately.

6. To check presence and integration of transgenes, extract total genomic DNA from young leaves of putative transgenic and non-transformed (control) plants by GenElute Plant Genomic DNA Miniprep Kit (Sigma). Perform PCR for presence of nptII and uidA genes using specifi c primers. To analyze the putative transformants by Southern blotting, digest the genomic DNA with restriction enzyme that has a single recog-nition site in the plasmid, resolve on 0.8 % agarose gel, transfer onto nylon membrane (Hybond N+, Amersham) using stan-dard protocol, and probe the blot with labeled (radioactive or nonradioactive) PCR-amplifi ed fragment of the nptII gene.

7. Analyze the progeny of self-pollinated transformants for the presence of uidA gene by PCR and RT-PCR. For RT-PCR, isolate total RNA from leaves of T 1 transformed (positive for uidA gene) and non-transformed control plants using RNeasy Plant Mini Kit (Qiagen, USA). Use 100 ng of total RNA and perform RT-PCR as per the instructions of the manufacturer using the Single-Step RT-PCR Kit (Qiagen, USA). Separate the amplifi ed products on 1 % agarose gel and stain with ethid-ium bromide to visualize the bands.

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Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224, DOI 10.1007/978-1-4939-1658-0_5, © Springer Science+Business Media New York 2015

Chapter 5