Recent advances in 3D bioprinting for cancer research: From precision models to personalized therapies.

Cancer remains one of the most devastating diseases, necessitating innovative and precise therapeutic solutions. The emergence of 3D bioprinting has revolutionized the platform of cancer therapy by offering bespoke solutions for drug screening, tumor modeling, and personalized medicine. The utilization of 3D bioprinting enables the fabrication of complex tumor models that closely mimic the in vivo microenvironment, facilitating more accurate drug testing and personalized treatment strategies. Moreover, 3D bioprinting also provides a platform for the development of implantable scaffolds as a therapeutic solution to cancer. In this review, we highlight the application of 3D bioprinting for cancer therapy along with current advancements in cancer 3D model development with recent case studies.

CRISPR-Cas for genome editing: Classification, mechanism, designing and applications.

Clustered regularly interspersed short pallindromic repeats (CRISPR) and CRISPR associated proteins (Cas) system (CRISPR-Cas) came into light as prokaryotic defence mechanism for adaptive immune response. CRISPR-Cas works by integrating short sequences of the target genome (spacers) into the CRISPR locus. The locus containing spacers interspersed repeats is further expressed into small guide CRISPR RNA (crRNA) which is then deployed by the Cas proteins to evade the target genome. Based on the Cas proteins CRISPR-Cas is classified according to polythetic system of classification. The characteristic of the CRISPR-Cas9 system to target DNA sequences using programmable RNAs has opened new arenas due to which today CRISPR-Cas has evolved as cutting end technique in the field of genome editing. Here, we discuss about the evolution of CRISPR, its classification and various Cas systems including the designing and molecular mechanism of CRISPR-Cas. Applications of CRISPR-Cas as a genome editing tools are also highlighted in the areas such as agriculture, and anticancer therapy. Briefly discuss the role of CRISPR and its Cas systems in the diagnosis of COVID-19 and its possible preventive measures. The challenges in existing CRISP-Cas technologies and their potential solutions are also discussed briefly.

Atmospheric cold plasma (ACP) treatment improved in-package shelf-life of strawberry fruit.

The aim of this study was to investigate the effect of atmospheric cold plasma (ACP) treatment on the microbial inactivation, physicochemical properties, and shelf-life of strawberry fruit with its extended in-package storage at room (25 °C) and refrigerated (4 °C) temperature. ACP treatment of 10, 15 and 30 min was studied on strawberry fruit using a dielectric barrier discharge (DBD) at 60 kV with an input voltage of 260 V at 50 Hz. The shelf-life of ACP treated strawberry was extended to 5 days at 25 °C and 9 days at 4 °C in sealed ACP package. However, non-treated packaged strawberry was degraded in 2 days. ACP treatment of 15 min resulted in 2 log reduction of microbial load and enhanced the concentration of chlorogenic acid, hyprin, phloretin, vanillin, gallic acid, 4-hydroxybenzaldehyde and rutin during in-package storage of 5 day (~ 120 h) at 25 °C with respect to control (p < 0.05). In addition, ACP treatment of 15 min at 60 kV was also found to increase the total phenolic content and antioxidant activity. However, total soluble solids, pH and moisture were not affected with ACP treatment (p > 0.05). Therefore, ACP treatment of 15 min with in-package storage of 5 days (~ 120 h) was found to be advantageous for increasing the shelf-life and functional quality of strawberry fruit.

Impact of atmospheric non-thermal plasma and hydrothermal treatment on bioactive compounds and microbial inactivation of strawberry juice: A hurdle technology approach.

The aim of this study was to investigate the hurdle effect of combining atmospheric cold plasma (ACP) with hydrothermal treatment on ascorbic acid, individual polyphenolic compounds, total phenolic content, and microbial inactivation of strawberry juice. Strawberry juice was treated with ACP for 10 and 15 min using dielectric barrier discharge at 60 kV with the input voltage of 260 V. The ascorbic acid concentration was retained maximum only in ACP treatment followed by ACP + hydrothermal treatment. Furthermore, ACP treatment for 10 min coupled with hydrothermal treatment resulted in the higher concentration of gallic acid, epigallocatechin, phloretin, naringin, hyprin, and 4-O-caffeoylquinic acid with respect to control (p < 0.05). In addition, ACP treatment for 10 min at 60 kV in combination with hydrothermal treatment resulted in increased total phenolic content (p < 0.05). Moreover, a 2-log microbial reduction was found in processed strawberry juice with ACP coupled-hydrothermal treatment in comparison to control juice (p < 0.05). Therefore, ACP treatment of 10 min followed by hydrothermal treatment was found to be advantageous processing for strawberry juice to retain nutritional quality and decrease microbial load. Moreover, further optimization of ACP or hydrothermal processing with utilization of preservatives could be achieved for desired microbial inactivation for an industrial process.

In vivo diabetic wound healing potential of nanobiocomposites containing bamboo cellulose nanocrystals impregnated with silver nanoparticles.

In diabetes, hyperglycemic state immensely hinders the wound healing. Here, nanobiocomposites (NCs) developed by impregnation of in situ prepared silver nanoparticles in the matrix of bamboo cellulose nanocrystals were investigated for their ability to hasten the progress of healing events in streptozotocin induced diabetic mice model. Wounds treated with topically applied NCs (hydrogels) showed full recovery (98-100%) within 18days post wounding in contrast to the various control groups where incomplete healing (88-92%) was noticed. Biochemical estimations documented a marked decrease in the levels of pro-inflammatory cytokines IL-6 and TNF-α leading to decreased inflammation in NCs treated mice. Significantly increased expression of collagen and growth factors (FGF, PDGF, VEGF) upon NCs treatment resulted in improved re-epithelialization, vasculogenesis and collagen deposition as compared to control groups. Hence, developed nanobiocomposites showcased potential to serve as highly effective and biocompatible wound dressings for diabetic patients.

Pyramiding of tea Dihydroflavonol reductase and Anthocyanidin reductase increases flavan-3-ols and improves protective ability under stress conditions in tobacco.

Tea (Camellia sinensis) is one of the richest sources of flavan-3-ols, an important class of flavonoids. The expression level of gene-encoded key regulatory enzymes of flavan-3-ol/anthocyanin biosynthetic pathway, dihydroflavonol 4-reductase (DFR) and anthocyanidin reductase (ANR), has been highly correlated with the flavan-3-ol contents and antioxidant activity in tea plant. In the present study, pyramiding of CsDFR and CsANR in tobacco was achieved. However, single transgenic tobacco overexpressing either CsDFR or CsANR was documented earlier. In continuation, pyramided transgenic lines were evaluated for the possible, either same or beyond, effect on flavan-3-ol accumulation and protective ability against biotic and abiotic stresses. The pyramided transgenic lines showed early flowering and improved seed yield. The transcript levels of flavan-3-ol/anthocyanin biosynthetic pathway and related genes in pyramided transgenic lines were upregulated as compared to control tobacco plants. The accumulations of flavan-3-ols were also found to be higher in pyramided transgenic lines than control tobacco plants. In contrast, anthocyanin content was observed to be decreased in pyramided transgenic lines, while DPPH activity was higher in pyramided transgenic lines. In pyramided transgenic lines, strong protective ability against feeding by Spodoptera litura was documented. The seeds of pyramided transgenic lines were also found to have better germination rate under aluminum toxicity as compared to control tobacco plants. Interestingly, the synergistic effect of these two selected genes are not beyond from transgenic lines expressing either CsDFR and CsANR alone as published earlier in terms of flavan-3-ols accumulation. However, the unique flower color and better seed germination rate are some interestingly comparable differences that were reported in pyramided lines in relation to individual transgenic plants. In conclusion, the present results reveal an interesting dynamic between CsDFR and CsANR in modulating flavan-3-ol/anthocyanin levels and functional analysis of stacked CsDFR and CsANR transgenic tobacco lines.

Anthocyanins enriched purple tea exhibits antioxidant, immunostimulatory and anticancer activities.

Purple coloured tea shoot clones have gained interest due to high content of anthocyanins in addition to catechins. Transcript expression of genes encoding anthocyanidin reductase (ANR), dihydroflavonol-4-reductase (DFR), anthocyanidin synthase (ANS), flavonol synthase (FLS) and leucoantho cyanidin reductase (LAR) enzymes in three new purple shoot tea clones compared with normal tea clone showed higher expression of CsDFR, CsANR, CsANS and lower expression of CsFLS and CsLAR in purple shoot clones compared to normal clone. Expression pattern supported high content of anthocyanins in purple tea. Four anthocyanins (AN1-4) were isolated and characterized by UPLC-ESI-QToF-MS/MS from IHBT 269 clone which recorded highest total anthocyanins content. Cyanidin-3-O-ß-d-(6-(E)-coumaroyl) glucopyranoside (AN2) showed highest in vitro antioxidant activity (IC50 DPPH = 25.27 ± 0.02 µg/mL and IC50 ABTS = 10.71 ± 0.01 µg/mL). Anticancer and immunostimulatory activities of cyanidin-3-glucoside (AN1), cyanidin-3-O-ß-d-(6-(E)-coumaroyl) glucopyranoside (AN2), delphinidin-3-O-ß-d-(6-(E)-coumaroyl) glucopyranoside (AN3), cyanidin-3-O-(2-O-ß-xylopyranosyl-6-O-acetyl)-ß-glucopyranoside (AN4) and crude anthocyanin extract (AN5) showed high therapeutic perspective. Anthocyanins AN1-4 and crude extract AN5 showed cytotoxicity on C-6 cancer cells and high relative fluorescence units (RFU) at 200 µg/mL suggesting promising apoptosis induction activity as well as influential immunostimulatory potential. Observations demonstrate potential of purple anthocyanins enriched tea clone for exploitation as a nutraceutical product.

PLA nanovectors with encapsulated betulin: plant leaf extract-synthesized nanovectors are more efficacious than PVA-synthesized nanovectors.

OBJECTIVES: Betulin (BT) is an abundant triterpene found predominantly in the bark of Himalayan birch. It is difficult to deliver it in vivo because of its low aqueous solubility. We have therefore developed novel formulations of BT for improving its solubility, bioavailability and therapeutic efficacy. RESULTS: Poly-D,L-lactide nanovectors (PLA NVs) were synthesized using poly(vinyl alcohol) and Lonicera japonica leaf extract (LE) as a stabiliser and named as PLA-1 NVs and PLA-2 NVs. PLA-1 NVs and PLA-2 NVs were used for the encapsulation of betulin (BT) and named as BT-En-1 and BT-En-2 NVs. The encapsulation efficiency of BT-En-1 and BT-En-2 NVs were 99.3 and 100 % respectively. Prepared nanoformulations were physically stable. An in vitro study revealed 45 % BT was released over 24 h. BT had a prolonged release from BT-En-2 NVs as compared to BT-En-1 NVs. BT-En-2 NVs had better anticancerous activity against SiHa cells than BT-En-1 NVs. CONCLUSIONS: Developed BT-EN-2 NVs had better biocompatibility, excellent stability and enhanced release characteristics than BT-En-1 NVs.

AtROS1 overexpression provides evidence for epigenetic regulation of genes encoding enzymes of flavonoid biosynthesis and antioxidant pathways during salt stress in transgenic tobacco.

In plants, epigenetic changes have been identified as regulators of developmental events during normal growth as well as environmental stress exposures. Flavonoid biosynthetic and antioxidant pathways play a significant role in plant defence during their exposure to environmental cues. The aim of this study was to unravel whether genes encoding enzymes of flavonoid biosynthetic and antioxidant pathways are under epigenetic regulation, particularly DNA methylation, during salt stress. For this, a repressor of silencing from Arabidopsis, AtROS1, was overexpressed in transgenic tobacco. Generated transgenics were evaluated to examine the influence of AtROS1 on methylation status of promoters as well as on coding regions of genes encoding enzymes of flavonoids biosynthesis and antioxidant pathways. Overexpression of AtROS1 increases the demethylation levels of both promoters as well as coding regions of genes encoding chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, flavonol synthase, dihydroflavonol 4-reductase, and anthocyanidin synthase of the flavonoid biosynthetic pathway, and glutathione S-transferase, ascorbate peroxidase, glutathione peroxidase, and glutathione reductase of the antioxidant pathway during control conditions. The level of demethylation was further increased at promoters as well as coding regions of these genes during salt-stress conditions. Transgenic tobacco overexpressing AtROS1 showed tolerance to salt stress that could have been due to the higher expression levels of the genes encoding enzymes of the flavonoid biosynthetic and antioxidant pathways. This is the first comprehensive study documenting the epigenetic regulation of flavonoid biosynthetic and antioxidant pathways during salt-stress exposure of plants.

Overexpression of a tea flavanone 3-hydroxylase gene confers tolerance to salt stress and Alternaria solani in transgenic tobacco.

Flavan-3-ols are the major flavonoids present in tea (Camellia sinensis) leaves. These are known to have antioxidant and free radical scavenging properties in vitro. Flavanone 3-hydroxylase is considered to be an important enzyme of flavonoid pathway leading to accumulation of flavan-3-ols in tea. Expression analysis revealed the upregulation in transcript levels of C. sinensis flavanone 3-hydroxylase (CsF3H) encoding gene under salt stress. In this study, the biotechnological potential of CsF3H was evaluated by gene overexpression in tobacco (Nicotiana tabacum cv. Xanthi). Overexpression of CsF3H cDNA increased the content of flavan-3-ols in tobacco and conferred tolerance to salt stress and fungus Alternaria solani infection. Transgenic tobaccos were observed for increase in primary root length, number of lateral roots, chlorophyll content, antioxidant enzyme expression and their activities. Also, they showed lesser malondialdehyde content and electrolyte leakage compared to control tobacco plants. Further, transgenic plants produced higher degree of pectin methyl esterification via decreasing pectin methyl esterase (PME) activity in roots and leaves under unstressed and salt stressed conditions. The effect of flavan-3-ols on pectin methyl esterification under salt stressed conditions was further validated through in vitro experiments in which non-transgenic (wild) tobacco seedlings were exposed to salt stress in presence of flavan-3-ols, epicatechin and epigallocatechin. The in vitro exposed seedlings showed similar trend of increase in pectin methyl esterification through decreasing PME activity as observed in CsF3H transgenic lines. Taken together, overexpression of CsF3H provided tolerance to salt stress and fungus A. solani infection to transgenic tobacco through improved antioxidant system and enhanced pectin methyl esterification.

Biosurfactant stabilized anticancer biomolecule-loaded poly (D,L-lactide) nanoparticles.

PLA nanoparticles (NPs) were prepared via green route using turmeric (Curcuma longa) extract (TE) as biostabiliser/biosurfactant. Of the 29 formulations, two formulations of TE synthesized PLA NPs were evaluated for encapsulation and controlled release of well known antioxidant and less bioavailable molecules curcumin and quercetin. Size of curcumin loaded PLA NPs synthesized using 0.8 mg/ml PLA (C-En-D) and 0.1 mg/ml PLA (C-En-P) were 203±77 and 110±44 nm, respectively. However, quercetin loaded PLA NPs synthesized at 0.8 mg/ml (Q-En-D) and 0.1mg/ml (Q-En-P) PLA concentrations were 170±95 and 102±31 nm, respectively. The encapsulation efficiency of curcumin loaded PLA (C-En-D and C-En-P) NPs as well as quercetin loaded PLA (Q-En-D and Q-En-P) NPs was found ∼95%. In vitro release study of C-En-D, C-En-P, Q-En-D and Q-En-P NPs showed initial burst release followed by slow and sustained release. C-En-D NPs and Q-En-D NPs showing maximum in vitro release (∼100%) were evaluated for cytotoxicity. Blank PLA NPs, C-En-D and Q-En-D NPs were found to be safe against normal human leukocytes up to 2 mg/ml dose. Both C-En-D and Q-En-D NPs showed anticancer activity against A549 cell line. But Q-En-D NPs showed better anticancer activity than C-En-D NPS on A549 cells. While blank PLA NPs did not possess anticancer activity. TE extract stabilized PLA NPs were non-toxic, biocompatible and safe to normal human leukocytes. Such technology will be better, effective and safer in use for anticancer as well as other biological application.

Encapsulation of catechin and epicatechin on BSA NPS improved their stability and antioxidant potential.

Nanoencapsulation of antioxidant molecules on protein nanoparticles (NPs) could be an advanced approach for providing stable, better food nutraceuticals and anticancer drugs. The bioavailability and stability of catechin (CAT) and epicatechin (ECAT) were very poor. In the present study, the CAT and ECAT were loaded on bovine serum albumin (BSA) NPs following desolvation method. The transmission electron microscope (TEM) and atomic force microscope (AFM) recorded size of CAT-BSA NPs and ECAT-BSA NPs were 45 ± 5 nm and 48 ± 5 nm respectively. The encapsulation efficiency of CAT and ECAT on BSA NPs was found to be 60.5 and 54.5 % respectively. CAT-BSA NPs and ECAT-BSA NPs show slow and sustained in vitro release. The CAT-BSA NPs and ECAT-BSA NPs were stable in solution at various temperatures 37 °C, 47 °C and 57 °C. DPPH assay revealed that CAT and ECAT maintained their functional activity even after encapsulation on BSA NPs. Furthermore, the efficacy of CAT-BSA NPs and ECAT-BSA NPs determined against A549 cell lines was found to be improved. CAT and ECAT aptly encapsulated in BSA NPs, showed satisfactory sustained release, maintained antioxidant potential and found improved efficacy. This has thus suggested their more effective use in food and nutraceuticals as well as in medical field.

Encapsulation of podophyllotoxin and etoposide in biodegradable poly-D,L-lactide nanoparticles improved their anticancer activity.

To improve the efficacy podophyllotoxin (PODO) and etoposide (ETOPO) were encapsulated in poly-d,l-lactide nanoparticles (PLA NPs). The size of synthesised PODO-loaded PLA NPs and ETOPO-loaded PLA NPs was 100 ± 17 nm and 163 ± 20 nm and their encapsulation efficiency was 17 and 48%, respectively. In vitro release studies showed initial burst release followed by slow and sustained release. In vitro cytotoxicity of synthesised NPs was assessed using A549 and CHO-K1 cells. Blank PLA NPs did not show any toxicity. While PODO-loaded PLA NPs showed higher in vitro cytotoxicity in comparison to ETOPO-loaded PLA NPs against both cell lines. Also, the cytotoxicity of both PODO-loaded PLA NPs and ETOPO-loaded PLA NPs was higher compared to pure drugs. Hence, this study documents the improvement in efficacy of these molecules upon encapsulation in PLA NPs and could be an important strategy for better therapeutics.

Osmotin-expressing transgenic tea plants have improved stress tolerance and are of higher quality.

Drought is a major stress that affects the yield and quality of tea, a widely consumed beverage crop grown in more than 20 countries of the world. Therefore, osmotin gene-expressing transgenic tea plants produced using earlier optimized conditions were evaluated for their tolerance of drought stress and their quality. Improved tolerance of polyethylene glycol-induced water stress and faster recovery from stress were evident in transgenic lines compared with the normal phenotype. Significant improvements in growth under in-vitro conditions were also observed. Besides enhanced reactive oxygen species-scavenging enzyme activity, the transgenic lines contained significantly higher levels of flavan-3-ols and caffeine, key compounds that govern quality and commercial yield of the beverage. The selected transgenic lines have the potential to meet the demands of the tea industry for stress-tolerant plants with higher yield and quality. These traits of the transgenic lines can be effectively maintained for generations because tea is commercially cultivated through vegetative propagation only.

Gain of function mutation in tobacco MADS box promoter switch on the expression of flowering class B genes converting sepals to petals.

One mutant transgenic line displaying homeotic conversion of sepals to petals with other phenotypic aberrations was selected and characterized at molecular level. The increased transcript level of gene encoding anthocyanidin synthase and petal specific class B genes, GLOBOSA and DEFECIENS in sepals of mutant line may be responsible for its homeotic conversion to petaloid organs. While characterizing this mutant line for locus identification, T-DNA was found to be inserted in 3' untranslated region of promoter of class B MADS box gene, GLOBOSA. Here, CaMV 35S promoter of T-DNA might be deriving the expression of class B genes.

Transgenic Tobacco Overexpressing Tea cDNA Encoding Dihydroflavonol 4-Reductase and Anthocyanidin Reductase Induces Early Flowering and Provides Biotic Stress Tolerance.

Flavan-3-ols contribute significantly to flavonoid content of tea (Camellia sinensis L.). Dihydroflavonol 4-reductase (DFR) and anthocyanidin reductase (ANR) are known to be key regulatory enzymes of flavan-3-ols biosynthesis. In this study, we have generated the transgenic tobacco overexpressing individually tea cDNA CsDFR and CsANR encoding for DFR and ANR to evaluate their influence on developmental and protective abilities of plant against biotic stress. The transgenic lines of CsDFR and CsANR produced early flowering and better seed yield. Both types of transgenic tobacco showed higher content of flavonoids than control. Flavan-3-ols such as catechin, epicatechin and epicatechingallate were found to be increased in transgenic lines. The free radical scavenging activity of CsDFR and CsANR transgenic lines was improved. Oxidative stress was observed to induce lesser cell death in transgenic lines compared to control tobacco plants. Transgenic tobacco overexpressing CsDFR and CsANR also showed resistance against infestation by a tobacco leaf cutworm Spodoptera litura. Results suggested that the overexpression of CsDFR and CsANR cDNA in tobacco has improved flavonoids content and antioxidant potential. These attributes in transgenic tobacco have ultimately improved their growth and development, and biotic stress tolerance.

Gold nanoparticle exposure induces growth and yield enhancement in Arabidopsis thaliana.

Nanotechnology has the potential to revolutionize agriculture field. Towards this effort, carbon nanotubes have recently been reported to induce growth enhancement of tobacco cells. In this study, exposure to 24 nm size gold nanoparticles (GNPs) at 10 µg/ml concentration was found to enhance the total seed yield of Arabidopsis thaliana by 3 times over the control. In addition, 24 nm size GNP exposure at both 10 and 80 µg/ml concentrations has significantly improved seed germination rate, vegetative growth and free radical scavenging activity. A considerable correlation was found between expression of key plant regulatory molecules, microRNAs (miRs) and seed germination, growth and antioxidant potential of A. thaliana on GNP exposure. This is the first report showing GNPs as a promising tool to enhance seed yield of plants.

Influence of human lactoferrin expression on iron homeostasis, flavonoids, and antioxidants in transgenic tobacco.

This study was aimed at to check the influence of human lactoferrin (hLF) expression on iron homeostasis, flavonoids, and antioxidants in transgenic tobacco. Transgenic tobacco expressing hLF cDNA under the control of a CaMV 35S promoter was produced. The iron content as well as chlorophyll content of transgenic tobacco was lower compared to mock and untransformed wild plants. Interestingly, hLF transgenic tobacco showed higher level of transcript expression for genes related to iron content regulation like iron transporter and metal transporter. While expression of genes related to iron storage such as ferritin 1 and ferritin 2 was downregulated. The transcript expression of genes encoding antioxidant enzymes such as glutathione reductase, glutathione-S-transferase, ascorbate peroxidase, and catalase was downregulated in hLF transgenic tobacco compared to controls. Further, the transcript expression of two important genes encoding dihydroflavonol reductase (DFR) and phenylalanine ammonia lyase regulatory enzymes of flavonoid biosynthesis pathway was analyzed. The expression of DFR was found to be downregulated, while PAL expression was upregulated in hLF transgenic tobacco compared to mock and untransformed wild plant. Total phenolics, flavonoids, and proanthocyanidins contents were found to be higher in hLF transgenic tobacco than the mock and untransformed wild plant. Results suggest that hLF expression in transgenic tobacco leads to iron deficiency, downregulation of antioxidant enzymes, and increase in total flavonoids.

Overexpression of CsANR increased flavan-3-ols and decreased anthocyanins in transgenic tobacco.

Anthocyanins and flavan-3-ols are distributed widely in plants and synthesized by a common biosynthetic pathway. Anthocyanin reductase (ANR) represents branching-point enzyme of this pathway converting anthocyanidins to flavan-3-ols. Since tea contains highest amount of flavonoids, a cDNA encoding anthocyanin reductase from tea (CsANR) was overexpressed in transgenic tobacco to check the influence on anthocyanin and flavan-3-ols. The transgenic tobacco was confirmed by genomic PCR and expression of transgene was analyzed through semiquantitative PCR. Interestingly flowers of transgenic tobacco were light pink/white in color instead of dark pink in wild tobacco, documenting the decrease in anthocyanins content. Upon measurement, flower anthocyanin content was found to be lesser. While flavan-3-ols (epicatechin and epigallocatechin) contents were increased in leaf tissue of transgenic lines. The expressions of other endogenous flavonoid biosynthetic pathway genes in different floral parts (sepal, petal, stamen, and carpel) of CsANR overexpressing tobacco as well as wild tobacco were analyzed. The transcript levels of PAL and CHI genes were downregulated, while transcript levels of F3H, FLS, CHS, ANR1, and ANR2 genes were upregulated in all floral parts of CsANR transgenic plants compared to wild tobacco. The expressions of DFR and ANS genes were also spatially modulated in different floral parts due to overexpression of CsANR. Thus, CsANR overexpression increased flavan-3-ols and decreased anthocyanin content by modulating the expressions of various flavonoid biosynthetic pathway genes in flower of tobacco. These changes might be responsible for the observed pollen tube in the pollens of CsANR overexpressing transgenic tobacco when they were still in the anther before pollination.

Post-transcriptional silencing of flavonol synthase mRNA in tobacco leads to fruits with arrested seed set.

Flavonoids are synthesized by phenylpropanoid pathway. They are known to participate in large number of physiological and biochemical processes in plants. Parthenocarpy and male sterility has earlier been reported by silencing chalcone synthase (CHS) encoding gene. Silencing of CHS has blocked the synthesis of most of useful flavonoids including flavan-3-ols and flavonols. Also, these studies could not identify whether parthenocarpy/male sterility were due to lack of flavan-3-ols or flavonols or both. Flavonol synthase (FLS) is an important enzyme of flavonoid pathway that catalyzes the formation of flavonols. In this article, we propose a novel strategy towards the generation of seedless or less-seeded fruits by downregulation of flavonol biosynthesis in tobacco (Nicotiana tabacum cv Xanthi) through post-transcriptional gene silencing (PTGS) of FLS encoding mRNA. The FLS silenced lines were observed for 20-80% reduction in FLS encoding gene expression and 25-93% reduction in flavonol (quercetin) content. Interestingly, these FLS silenced tobacco lines also showed reduction in their anthocyanidins content. While the content of flavan-3-ols (catechin, epi-catechin and epi-gallocatechin) was found to be increased in FLS silenced lines. The delayed flowering in FLS silenced lines could be due to decrease in level of indole acetic acid (IAA) at apical region of their shoots. Furthermore, the pollen germination was hampered and pollens were unable to produce functional pollen tube in FLS silenced tobacco lines. Pods of FLS silenced lines contained significantly less number of seeds. The in vitro and in vivo studies where 1 µM quercetin was supplied to germination media, documented the restoration of normal pollen germination and pollen tube growth. This finding identified the role of flavonols particularly quercetin in pollen germination as well as in the regulation of plant fertility. Results also suggest a novel approach towards generation of seedless/less-seeded fruits via PTGS of FLS encoding gene in plants.