In planta transformation in wheat: an improved protocol to develop wheat transformants.

BACKGROUND: Lack of efficient transformation protocol continues to be a major bottleneck for successful genome editing or transgenic development in wheat. An in planta transformation method was developed in Indian bread wheat in earlier study (Vasil et al. in Nat Biotechnol 10:667-674, 1992) which was labour-intensive and time-consuming. In the present study, in planta transformation method was improved to make it simple, efficient, less labour-intensive and time-saving. METHODS AND RESULTS: PCR-based screening for generated transformants at T0 stage was introduced in this method. Shoot apical meristem of two days old wheat seedling was inoculated with the routine active culture of Agrobacterium tumefaciens harboring plasmid pCAMBIA1300-Ubi-GFP having gene GFP under the control of Zea mays ubiquitin promoter. PCR analysis at T0 stage confirmed 27 plants to be transgene positive. These 27 plants were only taken to the next generation (T1) and the rest were discarded. At T1 generation 6 plants were analyzed to be PCR positive. Out of them, 4 plants were confirmed to have stable integration of transgene (GFP). Fluorescent microscopy at T1 stage confirmed the 4 Southern hybridization positive plants to be expressing reporter gene GFP. CONCLUSIONS: Screening at T0 stage, reduced the load of plants to be taken to T1 generation and their screening thereof at T1 with no overall loss in transformation efficiency. We successfully transformed wheat genotype HD2894 with 3.33% transformation efficiency using a simple, effective method which was less labour-intensive and less time-consuming. This method may be utilized to develop wheat transgenic as well as genome edited lines for desirable traits.

Influence of super-optimal light intensity on the acetic acid uptake and microalgal growth in mixotrophic culture of Chlorella sorokiniana in bubble-column photobioreactors.

This study seeks to determine the influence of super-optimal light intensity on acetic acid uptake and its associated impact on the cellular composition of Chlorella sorokiniana in a semi-batch mixotrophic cultivation setup. Unicellular green microalga Chlorella sorokiniana is grown in a 1L bubble-column photobioreactor at light intensities from 6000 to 14,000 lx (≈81 to 189 µmol.photons.m-2.s-1). We find that microalgal acetic acid utilization reduces as illumination increases from an optimal 10,000 lx (≈135 µmol.photons.m-2.s-1) to a super-optimal zone (>10000 lx). This lowers microalgal growth (2.75 g/L) and acetic acid intake, which peak at 6 mL/L (10000 lx) and drop to 2 and 1 mL/L at 12,000 and 14,000 lx, respectively. Concurrently, the maximum lipid yield decreases from 0.66 g/L (10000 lx) to 0.54 g/L (12000 lx) and 0.42 g/L (14000 lx). Hence, super-optimal illumination not only disturbs phototrophy but also affects the heterotrophic component, creating an imbalance between the two.

Excited-State Dynamics of a Photoacid: A Potential Probe for Recognizing Transition from Lamellar to Nonlamellar Inverted Structures of Liposome based Nanocarriers.

Liposomes of a cationic lipid dioctadecyldimethylammonium bromide (DODAB) are efficient nanocarriers of nucleic acids. Incorporation of a neutral lipid monoolein (MO) in excess (xMO >0.5) changes the lamellar organization of DODAB liposomes into non-lamellar inverted structures of DODAB/MO liposomes facilitating nucleic acid delivery to cells. Photoexcitation of 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS), a photoacid, initiates an excited state proton transfer (ESPT) reaction in its protonated form (ROH*) generating the deprotonated anionic form (RO- *). The fluorescence intensity ratio (IROH* /IRO-* ) of these two forms is governed by the ESPT dynamics, and increases with increasing MO content (xMO ) in the cationic liposomes of DODAB. Transition from lamellar organization of DODAB liposomes into non-lamellar inverted structures of DODAB/MO liposomes, due to incorporation of MO (xMO ~0.7), is manifested by a significant increase of ESPT time (τPT ) and the time constant of wobbling motion (τW ) of HPTS. Thus, the lamellar organizations of DODAB or DODAB-rich (xMO 0.2) liposomes and the non-lamellar organizations of MO-rich (xMO ~0.7) liposomes are recognized by significantly different excited state dynamics of the photoacid.

A novel molecular reporter for probing protein DNA recognition: An optical spectroscopic and molecular modeling study.

A novel benzo[a]phenoxazine-based fluorescent dye LV2 has been employed as a molecular reporter to probe recognition of a linker histone protein H1 by calf-thymus DNA (DNA). Fluorescence lifetime of LV2 buried in the globular domain of H1 (∼2.1 ns) or in the minor groove of DNA (∼0.93 ns) increases significantly to 2.65 ns upon interaction of the cationic protein with DNA indicating formation of the H1-DNA complex. The rotational relaxation time of the fluorophore buried in the globular domain of H1 increases significantly from 2.2 ns to 8.54 ns in the presence of DNA manifesting the recognition of H1 by DNA leading to formation of the H1-DNA complex. Molecular docking and molecular dynamics (MD) simulations have shown that binding of LV2 is energetically most favourable in the interface of the H1-DNA complex than in the globular domain of H1 or in the minor groove of DNA. As a consequence, orientational relaxation of the LV2 is significantly hindered in the protein-DNA interface compared to H1 or DNA giving rise to a much longer rotational relaxation time (8.54 ns) in the H1-DNA complex relative to that in pure H1 (2.2 ns) or DNA (5.7 ns). Thus, via a significant change of fluorescence lifetime and rotational relaxation time, the benzo[a]phenoxazine-based fluorescent dye buried within the globular domain of the cationic protein, or within the minor groove of DNA, reports on recognition of H1 by DNA.

Expression of a Pennisetum glaucum gene DREB2A confers enhanced heat, drought and salinity tolerance in transgenic Arabidopsis.

BACKGROUND: Pearl millet (Pennisetum glaucum) is an essential cereal crop, whose growth and yield are not impacted by abiotic stresses, such as drought, heat, and cold. The DREB transcription factors (TF) are some of the largest groups of TFs in plants and play varied roles in plant stress response and signal transduction. METHODS AND RESULTS: In the present study, PgDREB2A gene encoding a DREB transcription factor in pearl millet was functionally characterized in Arabidopsis. DREB2A proteins contain a conserved domain that binds toethylene responsive element binding factors. Three different T1 transgenic lines overexpressing PgDREB2A gene were identified by Southern blot. Quantitative real-time polymerase chain reaction exhibited that PgDREB2A could be induced under drought conditions. As compared with the control, PgDREB2A overexpressing transgenic Arabidopsis showed increased rate of seed germination and root growth in transgenic lines under higher concentrations of mannitol, NaCl, ABA, heat and cold stress. Additionally, PgDREB2A transgenic lines showed enhanced durability after rehydration and tolerance to drought and salt stress was augmented with increased proline and reduced MDA build-up and diminishing water loss. CONCLUSIONS: Results from this study suggested that PgDREB2A as a transcription factor may improve endurance to various abiotic stresses and can be employed for developing crops tolerant to abiotic stresses.

Decoding the Kinetic Pathways toward a Lipid/DNA Complex of Alkyl Alcohol Cationic Lipids Formed in a Microfluidic Channel.

Complexes of cationic liposomes with DNA have emerged as promising nonviral vectors for delivering genetic information into cells for gene therapy. Kinetics of the liposome/DNA complex (lipoplex) formation on a millisecond time scale are studied by monitoring time evolution of fluorescence of 8-anilino-1-naphthalene sulfonic acid (ANS) and ethidium bromide (EtBr) in a continuous flow microfluidic channel coupled to a fluorescence microscope. The formation of lipoplexes between calf thymus DNA and liposomes based on two novel cationic lipids (Lip1810 and Lip1814) are found to follow a two-step process with kinetic constants for the Lip1814/DNA complex (k1 = 1120-1383 s-1, k2 = 0.227-1.45 s-1) being significantly different from those (k1 = 68.53-98.5 s-1, k2 = 32.3-60.19 s-1) corresponding to formation of the Lip1810/DNA complex. The kinetic pathway leading to the formation of Lip1814/DNA complex is diffusion-controlled whereas the formation of Lip1810/DNA complex occurs by a conformational rearrangement-controlled pathway. The observed difference in the kinetics of lipoplex formation likely originates from different structures of the lipid/DNA complexes.

Novel ASR isolated from drought stress responsive SSH library in pearl millet confers multiple abiotic stress tolerance in PgASR3 transgenic Arabidopsis.

A genomic resource of drought stress responsive genes/ESTs was generated using Suppression Subtractive Hybridization (SSH) approach in a drought stress tolerant Pennisetum glaucum genotype 841B. Fifty five days old plants were subjected to drought stress after withholding water for different time intervals (10 days, 15 days, 20 days and 25 days). A forward subtractive cDNA library was prepared from isolated RNA of leaf tissue. Differential gene expression under drought stress was validated for selected nine contigs by RT-qPCR. A transcript homologous to Setaria italica ASR3 upregulated under drought stress was isolated from genotype 841B and characterized. Heterologous expression of PgASR3 was validated in Arabidopsis and confirmed under multiple abiotic stress conditions. A total of four independent transgenic lines overexpressing gene PgASR3 were analyzed by Southern blot at T1 stage. For drought stress tolerance, three independent lines (T2 stage) were analyzed by biochemical and physiological assays at seedling stage. The growth rate (shoot and root length) of transgenic seedlings improved as compared to WT seedling under differenct abiotic stress conditions. The three transgenic lines were also validated for drought stress tolerance and RT-qPCR analysis, at maturity stage. Under drought stress conditions, the mature transgenic lines showed higher levels of RWC, chlorophyll and proline but lower levels of MDA as compared to WT plants. PgASR3 gene isolated and validated in this study can be utilized for developing abiotic stress tolerant crops.

Transgenic pigeonpea events expressing Cry1Ac and Cry2Aa exhibit resistance to Helicoverpa armigera.

KEY MESSAGE: Independent transgenic pigeonpea events were developed using two cry genes. Transgenic Cry2Aa-pigeonpea was established for the first time. Selected transgenic events demonstrated 100% mortality of Helicoverpa armigera in successive generations. Lepidopteran insect Helicoverpa armigera is the major yield constraint of food legume pigeonpea. The present study was aimed to develop H. armigera-resistant transgenic pigeonpea, selected on the basis of transgene expression and phenotyping. Agrobacterium tumefaciens-mediated transformation of embryonic axis explants of pigeonpea cv UPAS 120 was performed using two separate binary vectors carrying synthetic Bacillus thuringiensis insecticidal crystal protein genes, cry1Ac and cry2Aa. T0 transformants were selected on the basis of PCR and protein expression profile. T1 events were exclusively selected on the basis of expression and monogenic character for cry, validated through Western and Southern blot analyses, respectively. Independently transformed 12 Cry1Ac and 11 Cry2Aa single-copy events were developed. The level of Cry-protein expression in T1 transgenic events was 0.140-0.175% of total soluble protein. Expressed Cry1Ac and Cry2Aa proteins in transgenic pigeonpea exhibited significant weight loss of second-fourth instar larvae of H. armigera and ultimately 80-100% mortality in detached leaf bioassay. Selected Cry-transgenic pigeonpea events, established at T2 generation, inherited insect-resistant phenotype. Immunohistofluorescence localization in T3 plants demonstrated constitutive accumulation of Cry1Ac and Cry2Aa in leaf tissues of respective transgenic events. This study is the first report of transgenic pigeonpea development, where stable integration, effective expression and biological activity of two Cry proteins were demonstrated in subsequent three generations (T0, T1, and T2). These studies will contribute to biotechnological breeding programmes of pigeonpea for its genetic improvement.