Agriculture Reference
In-Depth Information
(i) Plating: Agrobacterium strains are inserted in the YM/YEP semi-solid me-
dium containing antibiotics for two to three days before the transformation.
(ii) Inoculation: Inoculation of Agrobacterium using the targeted explants (micro-
spores, protoplast isolation from microspore-derived cell suspension cultures,
anther or microspore-derived embryos/callus) with co-cultivation medium
and cultures are incubated at 26 °C for 2-3 days.
(iii) Disinfection of Agrobacterium and screening of transformed callus: Treated
explants are washed properly with Claforan (250 mg/l). Blotting the calli with
sterile filter paper and transferring the dry calli to the selected medium and
incubated in dark at 28 °C for 4-6 weeks (The callus are transferred to a fresh
medium after two weeks).
(iv) Regeneration and screening: Transformed embryoids/calluses are transferred
to regeneration medium and incubated at 28 °C for 2-3 weeks in light for
regeneration.
(v) Root and shoot development: To promote root and shoot development of re-
generated plantlets, they are transferred to the rooting medium.
(vi) Flow cytometry analysis: The ploidy level of transgenic plants is estimated by
flow cytometry. Nuclei are isolated from the young leaf. For the measuremet
procedure, a PARTEC Ploidy Analyser II (Partec, Münster, Germany) flow
cytometry equipped with the software (Partec, Münster, Germany) is used.
(vii) Transfer of plants to soil and colchicine treatment: For chromosome doubling,
regenerated plants (if haploid) with good roots are immersed in 0.2 % col-
chine and 2 % DMSO (dimethyl sulfoxide) for 6 h and transferred to soil.
(viii) Homozygosity test: Testing of doubled haploid (DH) transgenic plants is
done. Evaluation of stress tolerence is done and plants are grown in the field/
greenhouse for DHs seed collection.
2.2.3   Molecular Analysis of Transgenic Plants
Molecular analysis of transgens is followed by the protocol of Sambrook et al.
( 1989 ), Datta et al. ( 1997 ), Vashisht et al. ( 2005 ), Tuteja ( 2010 ). Some common
steps of molecular analysis of cereal crops are mentioned below.
(i) Confirmation of transgenic plants by PCR: Total genomic DNA is extracted
from the putatively transgenic plants and control of untransformed plants is
carried out using modified cetyltrimethylammonium bromide (CTAB) meth-
od (Tuteja 2010 ).
(ii) Southern blot analysis: For Southern blot analysis of transplastomic plants,
total cellular DNA is extracted from leaf based on the CTAB method. A 2 µg
total DNA is digested overnight with the restricted enzyme, separated on a
0.7 % agarose gel and blotted on to nylon membranes (Nytran N, Schleicher
& Schuell, Dassel, Germany). Then, hybridization is performed using the pro-
tocol of Datta et al. ( 1997 ) and Sambrook et al. ( 1989 ).
(iii) Northern blot analysis: Total RNA is isolated from the leaves of transgen-
ic plants as well as from the untransformed control. The subsequent RNA
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