Aqueous solution of L-glutamine at a concentration of 2.0 g litre-1 (Sigma, India) was autoclaved at 121°C and 15 psi for 20 and 40 minutes and lyophilized to afford GlA20 and GlA40, respectively. The same concentration of aqueous L-glutamine solution that was not autoclaved but filter sterilized served as control (GFs).
Chemical characterization of autoclaved products
GlA20, GlA40 and control were subjected to TLC in order to study the structural changes in L-glutamine due to thermal degradation by autoclaving. Samples spotted on TLC plates were run in solvent systems comprising of butanol:water:acetic acid:: 4:1:5 and n-propanol:2-propanol:water:: 4:3:2 followed by heating at 300°C in an oven for 15 minutes. Visual changes in color and Rf values of the spots were noted after spraying ninhydrin reagent on chromatographed TLC plate.
The autoclaved samples (GlA20 and GlA40) were also characterized by modern spectroscopic techniques i.e. NMR and ESI-MS. NMR spectra were recorded on a Bruker Avance-300 spectrometer. Mass spectra were recorded on QTOF-Micro of Waters Micromass.
Agrobacterium growth in response to autoclaved products
The engineered Agrobacterium tumefaciens strain, GV3101 containing the plasmid p35SGUSINT with the gus reporter and nptII selection marker genes was used. To fresh cultures of this strain harvested at an optical density (OD) of 0.6 at A600 nm, 2.0 g litre-1 of GlA20, GlA40 and GFs (as standardized in an earlier study) were added and grown overnight as shake cultures in 20 ml Yeast Mannitol Broth (YMB) in dark at 28°C and 150 rpm for 24 hrs. In another experiment, freshly revived cultures of A. tumefaciens was grown in liquid basal MS medium  containing either of 2.0 g l-1 GFs, GlA20, GlA40 maintained at different pH i.e., 5.2, 5.6, 5.9 and 7.0. Growth in response to 2.0 g l-1 5-oxo proline (purchased from Sigma, USA) in MS and YMB was also tested at these pHs. All the results were confirmed using 1.5% agar solidified YMB and MS medium.
For each of these experiments, cultures grown in medium free of GFs, GlA20, GlA40 or 5-oxo proline served as control. A minimum of three replicates per treatment were used. Growth in each case as represented by population density (i.e., a value = optical density (OD) at A600 nm × 1 × 109 cfu ml-1) was measured at regular 12 hr interval for 24 and 48 hrs of incubation in case of liquid and solid media, respectively.
Agrobacterium virulence (vir) gene induction assay
Vir gene induction by the autoclaved products was tested using an octopine-type Agrobacterium strain A348 harboring the pSM219 plasmid with lacZ under the control of virH promoter in trans to the wild-type pTiA6 plasmid [18, 19]. GlA20, GlA40 or GFs were added to overnight grown bacterial cultures in YMB. The reporter β-galactosidase activity was measured, and the results were expressed in specific units calculated as described [18–20]. The popular vir gene inducer AS (100 μM) was used as a positive control for its maximal vir gene induction ability.
Facilitation of Agrobacterium infection of plants by the autoclaved products of L-glutamine
The effect of GlA20, GlA40 or GFs on Agrobacterium infection of plants was tested using tender leaves of a number of plant species like Podophyllum hexandrum (Indian may apple), Aloe vera (aloe), Lavendula officinalis (lavender), Rosa sp. (wild rose), Malus domestica (apple) rootstock MM106, Dendrocalamus asper (bamboo), Cynodon dactylon (grass), Zea mays (maize), Oryza sativa
(wheat), Aurocaria (ornamental gymnosperm) and Dryopteris (fern). The leaves of Nicotiana tabacum (tobacco) served as control.
All the leaves were washed with Tween 20 and surface sterilized using 0.01% mercuric chloride for 5-10 min followed by thorough rinsing in sterile de-ionized water to remove all traces of mercuric chloride. The surface sterilized leaves were immersed for 10 min in fresh overnight grown culture of A. tumefaciens strain GV3101 and co-cultivated for 1, 2, 3, 5, 6 and 8 days at 28°C in dark after blotting off excess bacteria. Basal MS medium containing 2.0 g l-1 of either of GlA40, GlA20, 5-oxoproline and GFs was used for co-cultivation. After each co-cultivation period, cefotaxime at 1 g l-1 was used to wash the explants free of all residual Agrobacterium which were cultured on basal MS medium containing 5 μM TDZ and 10 μM NAA (MSC) at pH 5.6 under culture lab conditions for callus formation. In order to identify the optimal conditions, different pH, i.e., 5.2, 5.6, 5.9 and 7.0 of the co-cultivation medium containing 2.0 g l-1 of GlA40 were also tested.
In another experiment, leaves of different plant species (tobacco, Indian may apple, aloe, lavender, wild rose, apple rootstock MM106, bamboo, grass, maize, rice, aurocaria and fern) were also transformed using different densities of Agrobacterium equivalent to 1 × 107, 1 × 108, 1 × 109 and 1 × 1010 cfu ml-1 at A600 nm. These were then co-cultivated on medium supplemented with either of GFs, GlA40, GlA20 or 5-oxo proline at pH 5.6.
Agrobacterium infected explants co-cultivated on medium containing 100 μM AS but free of GFs, GlA40, GlA20 or 5-oxo proline supplements served as positive control. A minimum of five replicates with three leaves per plant species were taken for each experiment which were repeated at least thrice.
The histo-chemical assay of Jefferson et al.  was used to assess the success of transgene delivery into explants. After 1, 2, 3, 5, 6 and 8 days of co-cultivation, explants, treated (co-cultivated on media containing GFs, GlA40, GlA20 or 5-oxo proline at different pH) and control (co-cultivated on media containing 100 μM AS but free of either GlA20, GlA40, GFs or 5-oxo proline) were randomly selected and immersed in assay buffer containing 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide (GUS) followed by vacuum infiltration for 15 minutes. After an overnight incubation in dark at 37°C, the expression of gus reporter gene was scored as blue spots and/or sectors per leaf explant and photographed using a Sony Cybershot DSC-F-828 camera. GUS assay was also done for the leaf explants transformed using different densities of Agrobacterium followed by co-cultivation on medium containing GFs, GlA40, GlA20 or 5-oxo proline as well as control.
PCR confirmation of genetic transformation
Callus tissue formed on the leaf explants of different plant species transformed in the presence and absence of either GFs, GlA40, GlA20 or 5-oxo proline were selected on MSC containing 100 μg ml-1 kanamycin. As described by Doyle and Doyle , total genomic DNA was extracted from the kanamycin resistant calli (500 mg). These were PCR amplified using 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 2 min followed by further extension cycle of 7 min at 72°C using a programmable Stratagene Robocycler Gradient 40. The isolated genomic DNA (50 ng) was amplified using 200 μM dNTPs, 1.5 U Taq DNA polymerase and 10 pmol of forward and reverse primers i.e., 5'-GGTGGGAAAGCGCGTTACAAG-3' and 5'-TGGATCCCGGCATAGTTAAA-3', respectively (Bangalore Genei, India) designed so as to amplify a 490 bp fragment of the gus gene). While 50 pg of plasmid DNA served as positive control, DNA from untransformed tissues were used as negative controls. The amplified products were finally resolved on 1.2% agarose gel using a 0'GeneRuler™ 100 bp plus DNA ladder from Fermentas, Life Sciences. Plant species (eg. Podophyllum and Aurocaria) that failed to produce leaf callus were not subjected to PCR.
Genomic DNA was isolated from callus tissues of different plant species transformed in presence and absence of α-amino glutarimide. The isolated DNA (10 μg) were digested with HindIII and EcoRI (New England Biolabs Inc. USA) and resolved on 0.8% agarose gel along with a 0'GeneRuler™ 100 bp plus DNA ladder (Fermentas, Life Sciences). This was then blotted onto a nylon membrane (Hybond-N, Amersham Biosciences, Little Chalfont, Buckinghamshire, UK) and hybridized with PCR amplified plasmid gus gene probe labeled with Biotin using Biotin DecaLabel DNA Labeling kit, Fermentas, Life Sciences. The chromogenic substrate BCIP/NBT (nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate, toluidine salt) was used to detect the hybridization products of the biotinylated probe and the streptavidin-alkaline phosphatase conjugate as blue-purple colored bands.