GUS Histochemical Analysis Research Articles

Transcription factor TaNF-YB2 interacts with partners TaNF-YA7/YC7 and transcriptionally activates distinct stress-defensive genes to modulate drought tolerance in T. Aestivum

BackgroundDrought stress limits significantly the crop productivity. However, plants have evolved various strategies to cope with the drought conditions by adopting complex molecular, biochemical, and physiological mechanisms. Members of the nuclear factor Y (NF-Y) transcription factor (TF) family constitute one of the largest TF classes and are involved in plant responses to abiotic stresses.ResultsTaNF-YB2, a NY-YB subfamily gene in T. aestivum, was characterized in this study focusing on its role in mediating plant adaptation to drought stress. Yeast two-hybrid (Y-2 H), biomolecular fluoresence complementation (BiFC), and Co-immunoprecipitation (Co-IP) assays indicated that TaNF-YB2 interacts with the NF-YA member TaNF-YA7 and NF-YC family member TaNF-YC7, which constitutes a heterotrimer TaNF-YB2/TaNF-YA7/TaNF-YC7. The TaNF-YB2 transcripts are induced in roots and aerial tissues upon drought signaling; GUS histochemical staining analysis demonstrated the roles of cis-regulatory elements ABRE and MYB situated in TaNF-YB2 promoter to contribute to target gene response to drought. Transgene analysis on TaNF-YB2 confirmed its functions in regulating drought adaptation via modulating stomata movement, osmolyte biosynthesis, and reactive oxygen species (ROS) homeostasis. TaNF-YB2 possessed the abilities in transcriptionally activating TaP5CS2, the P5CS family gene involving proline biosynthesis and TaSOD1, TaCAT5, and TaPOD5, the genes encoding antioxidant enzymes. Positive correlations were found between yield and the TaNF-YB2 transcripts in a core panel constituting 45 wheat cultivars under drought condition, in which two types of major haplotypes including TaNF-YB2-Hap1 and -Hap2 were included, with the former conferring more TaNF-YB2 transcripts and stronger plant drought tolerance.ConclusionsTaNF-YB2 is transcriptional response to drought stress. It is an essential regulator in mediating plant drought adaptation by modulating the physiological processes associated with stomatal movement, osmolyte biosynthesis, and reactive oxygen species (ROS) homeostasis, depending on its role in transcriptionally regulating stress response genes. Our research deepens the understanding of plant drought stress underlying NF-Y TF family and provides gene resource in efforts for molecular breeding the drought-tolerant cultivars in T. aestivum.

Development of an Efficient and Reproducible Embryonic Axis-targeted Tissue Culture-based Transformation Protocol for Pigeonpea (Cajanus cajan)

Background: Tissue culture-based transformation approach poses significant challenges for genetic modification of recalcitrant pigeonpea due to poor regeneration efficiency. In this study, we present a comprehensive high-frequency regeneration and transformation protocol using embryonic axesas explants. Methods: Optimization of transformation parameters and culture conditions for different stages of adventitious shoot regeneration, including shoot induction, elongation and rooting, significantly enhanced transformation efficiency. Agrobacterium-mediated transformation was carried out using embryonic axes explants with pCAMBIA 2301 vector. Culturing of explants on MS basal media supplemented with BAP 2.5 in Preculture, BAP 2.5 in Selection Medium I, IAA 0.5, GA 0.5 and Zeatin 1 in Selection Medium II resulted in formation of numerous multiple shoots from primordia-forming calli at axillary bud regions. Result: Shooting efficiency was 82% and 77%, for cultivars, Pusa 992 and Asha respectively. Rooting efficiency was nearly 100% for both when cultured on half-strength MScontaining 15 g/l sucrose and 0.5 mg/l IAA. Regenerated plants exhibited a high level ofsurvival of 90% upon transplantation into pots. Successful gene integration and expression were confirmed through GUS histochemical and molecular analyses, with approximately 57% and 65% transformation efficiencies for cultivars Pusa 992 and Asha, respectively. This study presents a robust and reproducible tissue culture-based transformation system for pigeonpea, applicable across diverse genotypes, offering new possibilities for genetic improvement in this crucial legume crop.

Deeply functional identification of TCS1 alleles provides efficient technical paths for low-caffeine breeding of tea plants.

Caffeine is an important functional component in tea, which has the effect of excitement and nerve stimulation, but excessive intake can cause insomnia and dysphoria. Therefore, the production of tea with low-caffeine content can meet the consumption needs of certain people. Here, in addition to the previous alleles of the tea caffeine synthase (TCS1) gene, a new allele (TCS1h) from tea germplasms was identified. Results of in vitro activity analysis showed that TCS1h had both theobromine synthase (TS) and caffeine synthase (CS) activities. Site-directed mutagenesis experiments of TCS1a, TCS1c, and TCS1h demonstrated that apart from the 225th amino acid residue, the 269th amino acid also determined the CS activity. GUS histochemical analysis and dual-luciferase assay indicated the low promoter activity of TCS1e and TCS1f. In parallel, insertion and deletion mutations in large fragments of alleles and experiments of site-directed mutagenesis identified a key cis-acting element (G-box). Furthermore, it was found that the contents of purine alkaloids were related to the expression of corresponding functional genes and alleles, and the absence or presence and level of gene expression determined the content of purine alkaloids in tea plants to a certain extent. In summary, we concluded TCS1 alleles into three types with different functions and proposed a strategy to effectively enhance low-caffeine tea germplasms in breeding practices. This research provided an applicable technical avenue for accelerating the cultivation of specific low-caffeine tea plants.

Standardization of efficient regeneration protocol and agrobacterium mediated genetic transformation in RPBio-226 variety of rice (Oryza sativa)

Studies related to the genome engineering in plants require an efficient protocol with high frequency callus induction and regeneration. This present study was conducted at PRR Biotech laboratory, Hyderabad in the year 2020 and aimed to develop an efficient protocol to achieve a high frequency callus induction and genetic transformation of RPBio-226, a Samba Mahsuri rice variety. In-vitro callus induction was carried out using MS media supplemented with different concentrations of 2,4-D viz., 1.0, 1.5, 2.0, 2.5 mg/L singly and in combinations with BAP viz., 0.1, 0.2, 0.4, 0.6 mg/L. Shoot induction was carried out using MS media supplemented with BAP 1.0, 1.5, 2.0, 2.5 mg/L and Kinetin 0.5, 1.0, 1.5, 2.0 mg/L and the Best response observed with 2,4-D and BAP at 2.0 and 0.1 mg/L, respectively. Agrobacterium tumefaciens strain EHA105 harboring PBI 121 plasmid (binary vector) was used in the process of rice transformation. An experiment was planned to use various parameters viz. 0.1, 0.2, 0.3, 0.4 and 0.5 ODs of Agrobacterium cells at 600nm. Embryogenic calli were co-cultivated with Agrobacterium supplemented with different concentrations of acetosyringone (AS) (0, 50, 100 and 150 μM) and 100 μM was found to give the best response for transformation. After Agrobacterium infection, infected calli were selected on the selection media with kanamycin as selection agent. The best hormone for shoot induction was BAP at 1.5 mg/L and kinetin at 0.5 mg/L and highest number of roots was achieved with 0.1 mg/L NAA in combination with 0.2 mg/L IBA. Regenerated plantlets were successfully acclimatized on soilrite mix showing the highest survival rate after 4 weeks of acclimatization. The npt II and Gus specific primers were used for PCR to identify stably transformed progeny that survived kanamycin treatment. GUS Histochemical Analysis of Callus was also carried out for the confirmation of transformation which helps us to generate transgenic plants in future.

Mutation of NtNRAMP3 improves cadmium tolerance and its accumulation in tobacco leaves by regulating the subcellular distribution of cadmium

Cadmium (Cd) is a harmful element that affects plant growth and development. Genetic improvements could be applied for enhancing Cd tolerance and accumulation in plants. Here, a novel Cd stress-induced gene, NtNRAMP3, was identified in tobacco. We constructed two NtNRAMP3-knockout (KO) tobacco lines using the CRISPR/Cas9 system, which enhanced Cd tolerance and Cd accumulation in tobacco leaves compared with those in the wildtype (WT). Subcellular localization analysis suggested that NtNRAMP3 is a tonoplast protein and GUS (β-glucuronidase) histochemical analysis showed that NtNRAMP3 is highly expressed in the conductive tissue of leaves. NtNRAMP3-KO tobacco showed reduced Cd translation from vacuole to cytosol in leaves compared with the WT, and its vacuolar Cd concentration was significantly higher (20.78–22.81%) than that in the WT; in contrast, Cd concentration in the cytosol was reduced by 13.72–20.15%, preventing chlorophyll degradation and reducing reactive oxygen species accumulation in the leaves. Our findings demonstrate that NtNRAMP3 is involved in regulating Cd subcellular distribution (controlling Cd transport from vacuoles to the cytosol) and affects Cd tolerance and its accumulation in tobacco. This provides a key candidate gene to improve the phytoremediation efficiency of plants via genetic engineering.

A novel TcS allele conferring the high-theacrine and low-caffeine traits and having potential use in tea plant breeding.

Theacrine (1,3,7,9-tetramethyluric acid) is a natural product with remarkable pharmacological activities such as antidepressant, sedative and hypnotic activities, while caffeine (1,3,7-trimethylxanthine) has certain side effects to special populations. Hence, breeding tea plants with high theacrine and low caffeine will increase tea health benefits and promote consumption. In this study, we construct an F1 population by crossing 'Zhongcha 302' (theacrine-free) and a tea germplasm 'Ruyuan Kucha' (RY, theacrine-rich) to identify the causal gene for accumulating theacrine. The results showed that the content of theacrine was highly negatively correlated with caffeine (R2 > 0.9). Bulked segregant RNA sequencing analysis, molecular markers and gene expression analysis indicated that the theacrine synthase (TcS) gene was the candidate gene. The TcS was located in the nucleus and cytoplasm, and the theacrine can be detected in stably genetic transformed tobacco by feeding the substrate 1,3,7-trimethyluric acid. Moreover, an in vitro enzyme activity experiment revealed that the 241st amino acid residue was the key residue. Besides, we amplified the promoter region in several tea accessions with varied theacrine levels, and found a 234-bp deletion and a 271-bp insertion in RY. Both GUS histochemical analysis and dual-luciferase assay showed that TcS promoter activity in RY was relatively high. Lastly, we developed a molecular marker that is co-segregate with high-theacrine individuals in RY's offspring. These results demonstrate that the novel TcS allele in RY results in the high-theacrine and low-caffeine traits and the developed functional marker will facilitate the breeding of characteristic tea plants.

A Stress-Responsive CaM-Binding Transcription Factor, bZIP4, Confers Abiotic Stress Resistance in Arabidopsis

The basic leucine zipper (bZIP) transcription factors (TFs) function as regulators of many key developmental and physiological processes in all eukaryotes. In this study, we characterized the function of Arabidopsis bZIP4, a group S bZIP, whose function was not known. We confirmed that bZIP4 localizes to the nucleus and has DNA-binding affinity. By qRT-PCR and GUS histochemical analysis, we showed that bZIP4 is specifically expressed in root and that its expression is induced by abiotic stress and ABA. By phenotypic analysis, we demonstrated that the root length and the germination rate of bZIP4 overexpression (bZIP4-Ox) were significantly longer and higher than those of the WT and bZIP4-SRDX under higher salt and glucose concentrations, indicating that bZIP4-Ox is insensitive and tolerant to abiotic stress. Despite that, we found that bZIP4-Ox had enhanced expression of genes encoding protein phosphatases suppressing ABA responsiveness. We also confirmed that bZIP4 interacts with CaM1 and showed that its DNA-binding affinity is inhibited by interaction with CaM1. We propose a model in which the increased cytosolic calcium concentration under stress conditions activates CaM1 to bind bZIP4 to remove it from promoters of genes encoding ABA negative regulators, allowing the plants to operate on a typical ABA signaling pathway.

An Apical Meristem-Targeted in planta Transformation Method for the Development of Transgenics in Flax (Linum usitatissimum): Optimization and Validation.

Efficient regeneration of explants devoid of intrinsic somaclonal variations is a cardinal step in plant tissue culture, thus, a vital component of transgenic technology. However, recalcitrance of economically important crops to tissue culture-based organogenesis ensues a setback in the use of transgenesis in the genetic engineering of crop plants. The present study developed an optimized, genotype-independent, nonconventional tissue culture-independent in planta strategy for the genetic transformation of flax/linseed. This apical meristem-targeted in planta transformation protocol will accelerate value addition in the dual purpose industrially important but recalcitrant fiber crop flax/linseed. The study delineated optimization of Agrobacterium tumefaciens-mediated transformation and stable T-DNA (pCambia2301:GUS:nptII) integration in flax. It established successful use of a stringent soilrite-based screening in the presence of 30 mg/L kanamycin for the identification of putative transformants. The amenability, authenticity, and reproducibility of soilrite-based kanamycin screening were further verified at the molecular level by GUS histochemical analysis of T0 seedlings, GUS and nptII gene-specific PCR, genomic Southern hybridization for stable integration of T-DNA, and expression analysis of transgenes by sqRT-PCR. This method resulted in a screening efficiency of 6.05% in the presence of kanamycin, indicating amenability of in planta flax transformation. The strategy can be a promising tool for the successful development of transgenics in flax.

Development of an efficient in-planta Agrobacterium-mediated transformation method for Iranian purslane (Portulaca oleracea L.) using sonication and vacuum infiltration

Purslane (Portulaca oleracea L.), a valuable medicinal herb, is used as a source of pharmaceutical components such as flavonoids, alkaloids, fatty acids, terpenoids, and sterols. Regeneration of transgenic purslane plantlets from transformed cells is a time-consuming procedure and needs hard work. In this study, in-planta transformation of purslane, using sonication and vacuum infiltration, is reported. The purslane seeds were infected through Agrobacterium tumefaciens strain LBA4404 having the uidA gene on pBI121 vector. Effective selection of transformants was performed by supplementing MS media with 250 mg l−1 kanamycin. Several factors affecting the in-planta procedure including pre-culture duration, acetosyringone dose, sonication, and vacuum infiltration duration were investigated. The results demonstrated that the highest number of GUS-positive purslane plantlets was obtained when the pre-cultured seeds were sonicated and vacuum-infiltered for 2 min in agro-bacterial cell suspension, and then co-cultivated in MS media having 100 µM acetosyringone. The integration and expression of uidA gene in transgenic purslane was successfully corroborated by southern blot and GUS histochemical analyses.

Tissue-specific and stress-inducible promoters establish their suitability for containment of foreign gene(s) expression in transgenic potatoes.

The present study was conducted to determine efficiency of green tissue-specific (pRCA) and stress-inducible promoters (pRD29A) to express E. coli beta-glucuronidase (gusA) genein transgenic potatoes compared with constitutive promoter (35S CaMV). The promoter fragments were isolated from their original source and cloned upstream to gusA in pCAMBIA-1301 binary vector to develop plant expression constructs, i.e., pRCA-pCAMBIA and pRD29A-pCAMBIA. Agrobacterium strain GV2260 harboring recombinant plasmids were used to infect leaf discs and internodal explant of Lady Olympia cultivar. GUS histochemical analysis was performed at different stages to determine GUS activity in transgenic plants. To determine activity of stress-inducible promoter (pRD29A), transgenic plants were exposed to heat, drought and combination of both heat and drought stress. The real time (RT-qPCR) and GUS florimetric assays revealed that pRD29A promoter gets more activated under drought, heat and combination of both stresses. GUS expression levels were more than 10 folds high with pRD29A promoter compared to control. Likewise, the reduced transcripts levels of gusA gene under control of pRCA promoter were found in tuber/roots of transgenic plants compared to 35S promoter. GUS florimetric assays also showed decreased or no GUS expression in tubers. In conclusion, the results encourage the appropriate use of promoters to drive the expression of foreign gene(s) for the development of potato lines tolerant to biotic and abiotic stress while minimizing the risks of transgenic technology in potatoes.

The potential and efficacy of Allium sativum leaf lectin (ASAL) against sap-sucking insect pests of transgenic maize

The gene sequence encoding the mannose-binding homodimeric protein Allium sativum leaf agglutinin (ASAL) was introduced into maize inbred lines to achieve resistance against sap-sucking corn leaf aphids in transgenic lines. Maize transformants were generated after the co-cultivation of immature maize embryos with the Agrobacterium strain LB4404 harbouring a recombinant Ti-binary ASAL construct driven by the polyubiquitin promoter. The preliminary screening of maize transformants was performed via GUS histochemical analysis, and PCR and Southern blotting confirmed the insertion of the transgene and its stable integration in five transgenic maize lines. Transcript abundance was quantified by a quantitative real-time PCR assay, which revealed variable expression of the ASAL transgene among five transgenic maize lines. The highest mRNA expression of the ASAL gene was found in the A23 transgenic maize line, while the lowest expression was found in the AU1 transgenic maize line. In planta bioassays in the T1 progeny of the transgenic maize lines revealed high resistance against corn leaf aphids compared to the control non-transgenic line. The mortality of the infesting aphids (Rhopalosiphum maidi) was found to vary from 40 to 71% compared to that of the non-transgenic control maize line.

Construction and function of a root-specific promoter SRSP

The responsibility of root is absorbing water and nutrients, it is an important plant tissue, but easily to be affected by biotic and abiotic stresses, affecting crop growth and yield. The design of a synthetic root-specific promoter provides candidate promoters for the functional analysis and efficient expression of stress-related genes in crop roots. In this study, a synthetic root-specific module (pro-SRS) was designed using tandem four-copies of root specific cis-acting elements (OSE1ROOTNODULE, OSE2ROOTNODULE, SP8BFIBSP8AIB, and ROOTMOTIFAPOX1), and fused with minimal promoter from the CaMV 35S promoter to synthesize an artificially synthetic SRSP promoter. The SRSP promoter was cloned in pCAMBIA2300.1 by replacing CaMV 35S promoter so as to drive GUS expression. The constructs with SRSP promoter were transformed in tobacco by Agrobacterium-mediated method. SRSP promoter conferred root-specific expression in transgenic tobacco plants through Real-time PCR (RT-PCR) analysis and GUS histochemical staining analysis. It is indicated that the repeated arrangement of cis-acting elements can realize the expected function of the promoter. This study laid a theoretical foundation for the rational design of tissue-specific promoters.

An improved Agrobacterium-mediated transformation method for cotton (Gossypium hirsutum L. ‘KC3’) assisted by microinjection and sonication

An efficient and reproducible Agrobacterium tumefaciens-mediated transformation method was developed for Gossypium hirsutum L. ‘KC3’ using a shoot apex explant with the combination of microinjection and sonication. To optimize the transformation efficiency, preculture conditions, acetosyringone concentration, A. tumefaciens cell density, and precision microinjection with sonication were evaluated. A high-throughput A. tumefaciens-mediated genetic transformation method was developed using the uidA gene. The shoot apex explants were microinjected with an A. tumefaciens strain EHA105 carrying the uidA gene and co-cultivated for 3 d on mMS medium fortified with 100 mM acetosyringone. Among the different parameters and conditions tested, preculturing explants for 24 h, microinjecting three times with an A. tumefaciens cell suspension of OD600 nm = 0.6 containing 100 mM acetosyringone, and sonicating explants for 20 s produced the maximum transformation efficiency of 20.25%. Putative transformants were transferred to regeneration medium supplemented with 0.8 mg L−1 1-naphthaleneacetic acid, 2 mg L−1 thidiazuron, 16% (v/v) of an Oscillatoria acuminata extracellular extract, and 50 mg L−1 kanamycin. Initially, the transgenic shoots were evaluated by GUS histochemical analysis, and integration was confirmed by polymerase chain reaction and Southern blot analysis. Plantlets transferred from an artificial growth chamber to greenhouse conditions showed a higher survival rate of 90% compared to the directly transferred plantlets. This simple and efficient transformation system could be valuable to transfer any gene of interest into cotton with high transformation efficiency in a short time.

Development of an efficient protocol for Agrobacterium mediated transformation of some recalcitrant indica rice varieties

Rice is a staple food whose productivity is affected by multiple environmental stresses. The use of biotechnological tools is the best possibility to develop rice varieties with increased productivity, exceptional grain quality and resistance to various stress. Several parameters in the genetic transformation (co-cultivation period, acetosyringone concentration, temperature of the co-cultivation medium, antibiotic concentration etc.) of rice varieties—Shalimar Rice 1, Jhelum and K 332 were optimized for the first time using β-glucuronidase (GUS) gene as a reporter. The GUS expression in presence of 100 μM acetosyringone at pH 5.2 was more at 22 °C as compared to 28 °C in all the rice varieties. The selection medium containing 250 mg l−1 of cefotaxime and carbenicillin prevented Agrobacterium overgrowth and the use of 50 mg l−1 hygromycin resulted in killing of the untransformed calli. The regeneration medium containing MS B5 medium supplemented with 4% sucrose, 3 g l−1 phytagel, 2.5 mg l−1 BAP, 1 mg l−1 zeatin, 0.2 mg l−1 NAA and 0.5 mg l−1 TDZ allowed efficient regeneration of the rice calli. The overall regeneration frequency of Shalimar Rice 1, Jhelum and K 332 were 64, 75 and 77% respectively. Putative transformed plants were analyzed for the presence of the transgenes through GUS histochemical analyses and southern blotting. The present protocol provides an efficient and rapid embryogenic callus induction, transformation and regeneration system, which could be conveniently used for producing genetically modified plants and might help in the transformation of other related rice genotypes.

Overexpression of MusaMYB31, a R2R3 type MYB transcription factor gene indicate its role as a negative regulator of lignin biosynthesis in banana.

Lignin and polyphenols are important cellular components biosynthesized through phenylpropanoid pathway. Phenylpropanoid pathway in plants is regulated by some important transcription factors including R2R3 MYB transcription factors. In this study, we report the cloning and functional characterization of a banana R2R3-MYB transcription factor (MusaMYB31) by overexpression in transgenic banana plants and evaluated its potential role in regulating biosynthesis of lignin and polyphenols. Sequence analysis of MusaMYB31 indicated its clustering with members of subgroup 4 (Sg4) of R2R3MYB family which are well known for their role as repressors of lignin biosynthesis. Expression analysis indicated higher expression of MusaMYB31 in corm and root tissue, known for presence of highly lignified tissue than other organs of banana. Overexpression of MusaMYB31 in banana cultivar Rasthali was carried out and four transgenic lines were confirmed by GUS histochemical staining, PCR analysis and Southern blot. Histological and biochemical analysis suggested reduction of cell wall lignin in vascular elements of banana. Transgenic lines showed alteration in transcript levels of general phenylpropanoid pathway genes including lignin biosynthesis pathway genes. Reduction of total polyphenols content in transgenic lines was in line with the observation related to repression of general phenylpropanoid pathway genes. This study suggested the potential role of MusaMYB31 as repressor of lignin and polyphenols biosynthesis in banana.

Establishment of an efficient Agrobacterium-mediated genetic transformation system in Pelargonium graveolens: an important aromatic plant

Rose-scented geranium is an important aromatic herb, have eminent for oil. The oil of geranium commercially utilized in the perfumery, cosmetic and aromatherapy industries all over the world. It is also helpful to cure many of the diseases, since it possess antibacterial, antifungal, antioxidant, anti-inflammatory and anticancer activities. However rose scented geranium suffer from several biotic and abiotic stresses, which reduced the yield of oil. So there is need to genetically improve the geranium using biotechnological approaches. The present study demonstrates the establishment of direct regeneration and Agrobacterium tumefaciens (LBA4404) mediated transformation protocol in Pelargonium graveolens (cv. CIM-BIO 171). Different media combinations such as benzyl amino purine (BAP), kinetin, naphthalene acetic acid (NAA), and adenine di-sulphate (ADS) were standardised to induce direct regeneration in P. graveolens. The maximum regeneration frequency i.e. 90.56 ± 1.2% per explant was achieved from petiolar segments in medium containing 2.5 mg/l BAP, 0.1 mg/l NAA, 1 mg/l ADS. However, with the leaf explants only 45.94 ± 2.91% frequency was achieved. In the present study, A. tumefaciens strain LBA4404 was used carrying binary vector pBI121 with the gusA as a reporter gene and neomycin phosphotransferase II (nptII) gene as a plant selectable marker. Parameters like bacterial optical density, infection time, acetosyringone concentration and kanamycin concentration were optimised to achieve maximum transformation frequency (69.5 ± 2.3%).The putative transgenic shoots were subsequently rooted on half strength MS medium and successfully transferred to the greenhouse. The transgenic plants were characterised by gus histochemical assay, PCR analysis (nptII-786 bp and gus A- 1707 bp) and Southern hybridization tests using gusA gene probe. The regeneration as well as transformation protocol will no doubt provide the basis to decipher the insights of metabolic pathways in geranium. Also could be useful for genetic improvement, to make it more tolerant/resistant against biotic and abiotic stresses and ultimately fruitful for Indian farmers in agronomic traits like high biomass, oil content, yield and better quality.

Assessment of factors influencing the tissue culture-independent Agrobacterium-mediated in planta genetic transformation of okra [Abelmoschus esculentus (L.) Moench

Regeneration of transformed plants from the Agrobacterium-infected tissue is a time-consuming process and requires hard work. Okra [Abelmoschus esculentus (L.) Moench] is highly recalcitrant to Agrobacterium-mediated genetic transformation and regeneration. In this study, we established a tissue culture-independent genetic transformation system for okra using seed as an explant. Agrobacterium tumefaciens EHA 105 harbouring the binary vector pCAMBIA 1301–bar was used to infect the okra seeds. Various parameters influencing the okra genetic transformation including, co-cultivation duration, acetosyringone, sonication, and vacuum infiltration have been evaluated. Maximum transformation efficiency of 18.3 % was recorded when the pre-cultured okra seeds were sonicated for 30 min and vacuum infiltrated for 3 min in Agrobacterium suspension containing 100 µM acetosyringone and co-cultivated for 3 days on a medium containing 100 µM acetosyringone. The GUS histochemical analysis confirmed the gus A gene integration and expression, whereas polymerase chain reaction (PCR) and Southern blot hybridization confirmed the bar gene integration and copy number in the transformed okra genome. The transgene was successfully segregated into the progeny plants with a Mendelian inheritance ratio of 3:1. The in planta transformation protocol developed in the present investigation is applicable to transform the okra plants with disease-resistant traits, and the transformed plants can be generated within 60 days.

Agrobacterium-mediated PsCBL and PsCIPK gene transformation to enhance salt tolerance in indica rice (Oryza sativa)

Salt tolerant crops are essential to bring the currently uncultivable salinity affected areas under cultivation. Here, we report Agrobacterium-mediated transformation of Pisum sativum calcineurin B-like protein (PsCBL) and P. sativum calcineurin B-like (CBL)-interacting protein kinase (PsCIPK) genes for development of salt-tolerant rice (Oryza sativa L.). The four indica rice cultivars used in this study were ‘BR 11’, ‘BRRI dhan 49’, ‘Paijam’, and ‘Chinigura’, with the maximum callus transformation frequency reaching 66.6 ± 7.2, 5.1 ± 6.2%, 79.1 ± 7.2 and 58.3 ± 7.2%, respectively. Among the transformed calli, the highest rates of regenerated plantlets were 62.9 ± 8.2% for ‘BR 11’, 73.5 ± 7.8% for ‘BRRI dhan 49’, and 65.5 ± 7.8% for ‘Chinigura’. ‘Paijam’ calli were recalcitrant to regeneration, which led us to directly transform ‘Paijam’ seeds without an intervening callus phase. The maximum transformation rate of ‘Paijam’ plantlets obtained was 46.3 ± 5.3%. Successful transformation events were confirmed through GUS histochemical assays and PCR analyses of putative transgenic plants. The transgenic rice plants ‘BR 11’, ‘BRRI dhan 49’, ‘Paijam’, and ‘Chinigura’ were able to survive exposure to 150 mM NaCl stress for over 3 wk, while non-transformed control plants became bleached and died within 1 wk for the same treatment. Our results indicated that stable transformation of these rice cultivars either with PsCBL or PsCIPK significantly improved salt tolerance.

Enhancement of Agrobacterium-mediated transformation efficiency of model plant using quorum sensing molecule, N-3-oxo-octanoyl-l-homoserine-lactone

Limitations of genetic transformation through Agrobacterium tumefaciens infection have been reported in many plant species, mainly due to low virulence of Agrobacterium to plant host and inefficient integration of T-DNA into plant genome. In this study, we investigated the effect of quorum sensing molecules, N-acylhomoserine-lactones (AHLs), on Agrobacterium-mediated transformation of leaf explants of Nicotiana tabacum cv. SR1. Our results revealed that AHLs significantly influenced the transformation frequency in N. tabacum. The highest percentage of transformation (92.2 %) was recorded when leaf explants infected with Agrobacterium were co-cultivated for 3 days on Murashige and Skoog medium supplemented with 100 µM N-3-oxo-octanoyl-l-homoserine-lactone. The presence of reporter genes in regenerated plants was determined by GUS histochemical staining, gfp visualization and PCR analysis. Our results indicated the promotive effect of AHLs in facilitating the transfer of desirable genes into N. tabacum. This improved protocol could be applicable to various species for functional genomics studies and biotechnological applications including crop improvement programs.

Salt tolerance and activity of antioxidative enzymes of transgenic finger millet overexpressing a vacuolar H+-pyrophosphatase gene (SbVPPase) from Sorghum bicolor

A vacuolar proton pyrophosphatase cDNA clone was isolated from Sorghum bicolor (SbVPPase) using end-to-end gene-specific primer amplification. It showed 80–90% homology at the nucleotide and 85–95% homology at the amino acid level with other VPPases. The gene was introduced into expression vector pCAMBIA1301 under the control of the cauliflower mosaic virus 35S (CaMV35S) promoter and transformed into Agrobacterium tumifaciens strain LBA4404 to infect embryogenic calli of finger millet (Eleusine coracana). Successful transfer of SbVPPase was confirmed by a GUS histochemical assay and PCR analysis. Both, controls and transgenic plants were subjected to 100 and 200mM NaCl and certain biochemical and physiological parameters were studied. Relative water content (RWC), plant height, leaf expansion, finger length and width and grain weight were severely reduced (50–70%), and the flowering period was delayed by 20% in control plants compared to transgenic plants under salinity stress. With increasing salt stress, the proline and chlorophyll contents as well as the enzyme activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and glutathione reductase (GR) increased by 25–100% in transgenics, while malondialdehyde (MDA) showed a 2–4-fold decrease. The increased activities of antioxidant enzymes and the reduction in the MDA content suggest efficient scavenging of reactive oxygen species (ROS) in transgenics and, as a consequence, probably alleviation of salt stress. Also, the leaf tissues of the transgenics accumulated 1.5–2.5-fold higher Na+ and 0.4–0.8-fold higher K+ levels. Together, these results clearly demonstrate that overexpression of SbVPPase in transgenic finger millet enhances the plant's performance under salt stress.