Efficient Agrobacterium mediated genetic transformation of CRISPR/Cas9 construct using cotyledonary node explants of greengram

An efficient protocol for generating transformed greengram plants by choosing prominent explant which is used to generate transformed plantlets for their survivability and also with high transformation efficiency. However, the crop production is reduced due to factors. In current work, we employed transgenic free approach using CRISPR/Cas9 tool for establishing transformation protocol. The CRISPR/Cas9 is getting momentum widely for developing transgene free crops. In this approach, guide RNA encoding for ~18-23 bp sequence that contains a PAM (protospacer adjacent motif) on either strand of DNA, where CRISPR incepts with a single guide RNA (sgRNA) to target genes and initiate excision of a complimentary strands through the Cas9 endonuclease. Explants’ effects on CRISPR/Cas9 construct transformation were studied. For transformation, three distinct explants cotyledonary node (CN- MSB5 +3% sucrose+ 0.5 mg/L BAP for CN), shoot tip (ST- MSB5 + 3% sucrose+ + 0.5 mg/L BAP + 0.01 mg/L NAA), and immature cotyledon (IMC-3% sucrose + 0.5 mg/L BAP + 0.1 mg/L NAA) were used. The prepared explants were infected with EHA105 strain of Agrobacterium tumefaciens harboring a binary vector of pMDC100 containing npt II gene as screenable marker and Cas9. The explants were selected on 50 mg/L kanamycin medium. The transformed plants were confirmed by PCR using npt II and Cas9 specific primers. Cotyledonary node explant was found to be more efficient for CRISPR/Cas9 transformation and also it promisingly showed transgene integration through PCR analysis having more transformation efficiency and can be used for the production of transgenic free greengram crop using CRISPR/Cas9 approach

EVALUATION OF IN VITRO ANTICANCER AND ANTIOXIDANT ACTIVITIES FROM LEAF EXTRACTS OF MEDICINAL PLANT CLIDEMIA HIRTA

Objective: To evaluate the anticancer and antioxidant activity of medicinal plant Clidemia hirta extracted in different solvents.Methods: Crude extracts were prepared from the leaves of Clidemia hirta using ethanol, petroleum ether and chloroform solvents. Anticancer activities and antioxidant properties were assayed using standard yellow dye 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and 1, 1-diphenyl-2-picryl hydrazyl (DPPH) free radical scavenging assay respectively.Results: We found that the ethanol extract had higher inhibition activities against Dalton's lymphoma ascites (DLA) cancer cell line, 50% DLA cell line inhibition at 68µg/ml, while 50% inhibition by petroleum ether and chloroform extracts were at 160µg/ml and 172µg/ml, respectively. The antioxidant activity requires5µg/ml of ethanol extract to trap 50% of DPPH (IC50), whereas the positive control ascorbic acid trapped 50% of DPPH (IC50) at 3.5µg/ml.Conclusion: The prepared leaf extracts with different solvents of Clidemia hirta showed the antiproliferative and antioxidant activity in dose-dependent manner. Further works is required to identify the biologically active chemical constituents, responsible for cancer cell growth inhibition from this plant

<i>Cajanus platycarpus</i> Flavonoid 3′5′ Hydroxylase_2 (<i>CpF3′5′H_2</i>) Confers Resistance to <i>Helicoverpa armigera</i> by Modulating Total Polyphenols and Flavonoids in Transgenic Tobacco

Pod borer Helicoverpa armigera, a polyphagus herbivorous pest, tremendously incurs crop damage in economically important crops. This necessitates the identification and utility of novel genes for the control of the herbivore. The present study deals with the characterization of a flavonoid 3′5′ hydroxylase_2 (F3′5′H_2) from a pigeonpea wild relative Cajanus platycarpus, possessing a robust chemical resistance response to H. armigera. Though F3′5′H_2 displayed a dynamic expression pattern in both C. platycarpus (Cp) and the cultivated pigeonpea, Cajanus cajan (Cc) during continued herbivory, CpF3′5′H_2 showed a 4.6-fold increase vis a vis 3-fold in CcF3′5′H_2. Despite similar gene copy numbers in the two Cajanus spp., interesting genic and promoter sequence changes highlighted the stress responsiveness of CpF3′5′H_2. The relevance of CpF3′5′H_2 in H. armigera resistance was further validated in CpF3′5′H_2-overexpressed transgenic tobacco based on reduced leaf damage and increased larval mortality through an in vitro bioassay. As exciting maiden clues, CpF3′5′H_2 deterred herbivory in transgenic tobacco by increasing total flavonoids, polyphenols and reactive oxygen species (ROS) scavenging capacity. To the best of our knowledge, this is a maiden attempt ascertaining the role of F3′5′H_2 gene in the management of H. armigera. These interesting leads suggest the potential of this pivotal branch-point gene in biotic stress management programs

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Not AvailableIntensive cultivation, climate change and host–pathogen dynamics/interaction had led to emergence of viral diseases and vector population, thereby causing significant economic losses. In the present study, extensive surveys were conducted (during 2016–2017) followed with field experiments (2017 and 2019 kharif and 2018 rabi seasons) in the state of Chhattisgarh, India, comprising of three diverse agroecological zones, viz., northern hill zone, central plain zone and southern Bastar plateau zone, for understanding the distribution pattern of viruses and vector populations in prominent pulses and vegetable crops. Field experiments revealed that more than 90% incidence of yellow mosaic disease (YMD) was recorded in mungbean and urdbean, followed by YMD of ridge gourd and bitter gourd, leaf curl disease of tomato (ToLCD) and chilli and yellow vein mosaic disease of bhendi; however, only five per cent YMD was recorded in cow pea. Molecular and sequence analysis of viral genomic components revealed ToLCD in central plain zone of Chhattisgarh was caused by a consortium of begomoviruses, including Tomato leaf curl New Delhi virus (ToLCNDV), Tomato leaf curl Karnataka virus (ToLCKV), and Tomato leaf curl Kerala virus associated with satellite molecules. YMD of mungbean, cow pea and soybean was caused by Mungbean yellow mosaic India virus (MYMIV), while YMD of ridge gourd was caused by ToLCNDV. PCR analysis, using species-specific primers, of samples representing the three different regions revealed that YMD of mungbean, urdbean and cowpea was positive only for MYMIV and was unevenly distributed, while YMD of ridge gourd, bitter gourd, pumpkin and sponge gourd was positive only for ToLCNDV; however, ToLCD was positive for either ToLCNDV or ToLCKV or both, and some samples were also found associated with betasatellite molecules. Amaranthus and rice bean were identified as non-host species for the begomoviruses infecting tomato and MYMIV, as observed by surveys in the hot-spot-like areas, controlled whitefly transmission as well as agroinoculation. Compilation and analysis of available information on begomoviruses affecting these crops in India suggest that they are unevenly distributed in India. This is the first report of the begomovirus occurrence in pulses and vegetable crops in this agroecologically diverse region.Not Availabl