scholarly journals An Agrobacterium mediated transformation system of guava (Psidium guajava L.) with endochitinase gene

2014 ◽  
Vol 14 (4) ◽  
pp. 232-237 ◽  
Author(s):  
Maneesh Mishra ◽  
Syed Uzma Jalil ◽  
Nimisha Sharma ◽  
Umesh Hudedamani
Keyword(s):  
Transformation Efficiency ◽  
Binary Vector ◽  
Nuclear Genome ◽  
Psidium Guajava ◽  
Nptii Gene ◽  
Cultivation Medium ◽  
Putative Transformants ◽  
Selection Medium ◽  
First Time

Genetic transformation of guava (Psidium guajava L.) was developed for the first time using in vitro grown shoot tip explant co-cultivated with Agrobacterium tumefaciens strain LBA4404 harbouring binary vector pIIHR-JBMch with endochitinase and nptII genes. The highest transformation efficiency was achieved by wounding explants with tungsten particles (0.5 µm) through particle acceleration system, followed by infection for 45 minutes with A. tumefaciens, grown overnight with 100 µM acetosyringone, corresponding to OD600=0.5 followed by co-cultivation for 72 hours under dark condition on co-cultivation medium (MS+100 µM acetosyringone+100 mg L-1 L-Cystein). Putative transformed explants regenerated shoots on selection medium stressed with 200 mg L-1 kanamycin for 12 weeks. Molecular analysis of putative transformants by PCR confirmed the integration of endochitinase and nptII gene in the plant nuclear genome.

scholarly journals Agrobacterium-mediated Transformation of Cotyledon Explants of Bottle Gourd (Lagenaria siceraria S.)

HortScience ◽  
2004 ◽  
Vol 39 (4) ◽  
pp. 821E-822
Author(s):  
Jeung-Sul Han* ◽  
Chang Kil Kim
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Binary Vector ◽  
Bottle Gourd ◽  
Infection Frequency ◽  
Ms Medium ◽  
Lagenaria Siceraria ◽  
Cultivation Medium ◽  
Glufosinate Ammonium ◽  
Cotyledon Explants ◽  
Selection Medium

A procedure for producing transgenic bottle gourd plants by inoculating cotyledon explants with Agrobacterium tumefaciens strain AGL1 carrying a binary vector pCAMBIA3301, which contains glufosinate ammonium-resistant (bar) and the reporter (gus) genes, is describe. Infection was the most effective (highest infection frequency and index) when explants were co-cultivated with Agrobacterium for 6-8 days on co-cultivation medium supplemented with 0.001-0.1 mg/L L-a-(2-aminoethoxyvinyl) glycine (AVG). Transgenic plants were obtained with frequencies of about 0.2% when the explants were cultured on selection medium (MS medium supplemented with 3.0 mg/L BAP, 0.5 mg/L AgNO3, 500 mg/L cefotaxime, 2.0 mg/L DL-phosphinothricin, 0.3% sucrose and 0.8% Plant Agar. A histochemical gus assay, PCR and Southern blot analyses confirmed that transformation had occurred. Genetic analysis of T1 progenies showed that the transgenes were inherited in a Mendelian fashion. To our knowlege, this study represents the first report for Agrobacterium-mediated transformation in bottle gourd, rootstock for watermelon and other cucurbit crops in many countries.

scholarly journals Agrobacterium-mediated transformation of Citrus sinensis and Citrus limonia epicotyl segments

2003 ◽  
Vol 60 (1) ◽  
pp. 23-29 ◽  
Author(s):  
Weliton Antonio Bastos de Almeida ◽  
Francisco de Assis Alves Mourão Filho ◽  
Beatriz Madalena Januzzi Mendes ◽  
Alexandra Pavan ◽  
Adriana Pinheiro Martinelli Rodriguez
Keyword(s):  
Genetic Transformation ◽  
Citrus Sinensis ◽  
Transformation Efficiency ◽  
Petri Dish ◽  
Cultivation Medium ◽  
Breeding Programs ◽  
Rangpur Lime ◽  
Citrus Breeding ◽  
In Vitro Plantlets

Genetic transformation allows the release of improved cultivars with desirable characteristics in a shorter period of time and therefore may be useful in citrus breeding programs. The objective of this research was to establish a protocol for genetic transformation of Valencia and Natal sweet oranges (Citrus sinensis L. Osbeck) and Rangpur lime (Citrus limonia L. Osbeck). Epicotyl segments of germinated in vitro plantlets (three weeks in darkness and two weeks in a 16-h photoperiod) were used as explants. These were co-cultivated with Agrobacterium tumefaciens strain EHA-105 and different experiments were done to evaluate the transformation efficiency: explants were co-cultivated with Agrobacterium for one, three or five days; explants were incubated with Agrobacterium suspension for 5, 10, 20 or 40 minutes; co-cultivation medium was supplemented with acetosyringone at 0, 100 or 200 µmol L-1; Explants ends had a longitudinal terminal incision (2-3 mm); co-cultivation temperatures of 19, 23 or 27°C were imposed. The experimental design was completely randomized in all experiments with five replications, each consisted of a Petri dish (100 x 15 mm) with 30 explants and resulted in a total of 150 explants per treatment. Longitudinal terminal incision in the explant ends did not improve shoot regeneration. However, transgenic plants of all three cultivars were confirmed from explants that had been subjected to inoculation time of 20 minutes, co-culture of three days at 23-27°C, in the absence of acetosyringone.

scholarly journals Deciphering the role of fungal calmodulin gene using Host-mediated gene silencing approach in apple

2021 ◽  
Author(s):  
Shalini Verma ◽  
Manju Modgil ◽  
Arjun Chauhan
Keyword(s):  
Gene Silencing ◽  
Transgene Expression ◽  
Selective Medium ◽  
Pcr Analysis ◽  
Nptii Gene ◽  
Calmodulin Gene ◽  
Putative Transformants ◽  
Marssonina Coronaria

Abstract Premature leaf fall caused by Marssonina coronaria is one of the most destructive diseases of apple in India. In this study, host induced gene silencing approach was exploited to develop resistance to this disease in an apple cultivar ‘Red Chief’. Calmodulin gene (CaM) having its role in fungal differentiation, development and pathogenicity was selected as target. hpRNAi construct was prepared from the conserved off target free partial gene sequence of CaM and used for transformation trials. Upto 6% kanamycin resistant shoots were obtained on selective medium having 5–6 mg/l kan after 7 weeks of coculture. In PCR analysis of 13 RNAi putative transformants, 10 lines were found positive with CaM and nptII gene specific primers and six lines showed hybridization signal. Semi qRT-PCR revealed variable levels of transgene expression among RNAi lines which seems to be related to copy number of integrated gene. In vitro detached leaf assay revealed lesion development and disease progression in wild type after 5 dpi but not visible in five CaM RNAi lines. Microscopic examination of infected control leaves showed fully developed, septate mycelium, and conidia along with necrosis of whole tissue while three transformants showed reduced growth and differentiation of fungus and in rest three, hyphal development and necrosis were strongly restricted. We conclude that trafficking of dsRNA/ siRNA from apple plants to pathogen might have triggered the down regulation of fungal CaM gene which confirms that deciphering the role of CaM through HIGS lead to resistance to Marssonina blotch in apple.

scholarly journals REGENERATION AND TRANSFORMATION OF VFNT CHERRY TOMATO

HortScience ◽  
1990 ◽  
Vol 25 (9) ◽  
pp. 1078b-1078 ◽  
Author(s):  
J.M. Van Eck ◽  
E.D. Earle
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Chromosomal Location ◽  
Binary Vector ◽  
Culture Conditions ◽  
Pcr Analysis ◽  
Nptii Gene ◽  
Neomycin Phosphotransferase ◽  
Cherry Tomato ◽  
Number Of Shoots

Regeneration from VFNT Cherry tomato was optimized prior to transformation. Cotyledon and hypocotyl sections from 7-day-old in vitro grown VFNT Cherry tomato were cultured on medium containing MS salts, B5 vitamins and the following per liter: sucrose, 30g; zeatin, 1 mg; IAA, 0.1 mg; agar, 8.0g or GelriteR, 2.2g. Culture conditions investigated included cotyledon and upper hypocotyl explant lengths, light vs. dark germination, and agar vs. GelriteR. The conditions which resulted in the highest average number of shoots per explant were cotyledon basal explants 2 mm in length, 3.95; cotyledons from dark germination, 6.2; and hypocotyls from light germination, 8.6. An equal number of shoots regenerated on medium containing agar or GelriteR, however, shoots regenerated on medium containing agar were more vigorous. Cotyledon and hypocotyl sections were cocultivated with the Agrobacterium tumefaciens binary vector pBI121 containing the neomycin phosphotransferase II (NPTII) and B-glucuronidasc (GUS) genes. Transformants were selected by regeneration and rooting on medium containing kanamycin. Southern blot and PCR analysis indicated regeneranrs contain the NPTII and GUS genes. Mapping the chromosomal location of the NPTII gene is in progress.

scholarly journals Transformation of `Beurre Bosc' Pear with the rolC Gene

1999 ◽  
Vol 124 (6) ◽  
pp. 570-574 ◽  
Author(s):  
Richard L. Bell ◽  
Ralph Scorza ◽  
Chinnathambi Srinivasan ◽  
Kevin Webb
Keyword(s):  
Enzyme Activity ◽  
Agrobacterium Tumefaciens ◽  
Binary Vector ◽  
Leaf Explants ◽  
Rolc Gene ◽  
Proliferation Medium ◽  
Reduced Height ◽  
Kanamycin Selection ◽  
Selection Medium

`Beurre Bosc' pear (Pyrus communis L.) was transformed with Agrobacterium tumefaciens (E.F. Smith & Townsend) Conn strain EHA101 containing the binary vector pGA-GUSGF into which the rolC gene had been inserted. Leaf explants from in vitro shoot tip cultures were wounded, Agrobacterium-inoculated, and cultured on kanamycin selection medium. Regenerating shoots were transferred to proliferation medium without antibiotics. Three clones tested positive for GUS and nptII enzyme activity. Transformation with the rolC gene was confirmed by DNA, RNA, and protein blot analyses. The number of copies of the rolC transgene varied from one to three. Plantlets of the three transgenic clones were acclimated and transferred to the greenhouse. Preliminary observations of phenotype indicate that the rolC gene reduced height, number of nodes, and leaf area of transgenic `Beurre Bosc'.

In vitro antioxidant potential of spray-dried Psidium guajava L. leaves extracts

Planta Medica ◽  
2013 ◽  
Vol 79 (13) ◽  
Author(s):  
MR Fernandes ◽  
CR Souza ◽  
ML Martinez ◽  
WP Oliveira
Keyword(s):  
Psidium Guajava ◽  
Antioxidant Potential ◽  
Spray Dried

Somatic Embryogenesis and In vitro Plant Regeneration in Vigna trilobata (L.) Verd. - A Potential Pasture Legume

1970 ◽  
Vol 19 (1) ◽  
pp. 89-99
Author(s):  
K. Choudhary ◽  
M. Singh ◽  
M. S. Rathore ◽  
N. S. Shekhawat
Keyword(s):  
Somatic Embryogenesis ◽  
Growth Regulators ◽  
Somatic Embryos ◽  
Basal Medium ◽  
Long Term Study ◽  
Continuous Application ◽  
First Time ◽  
Pasture Legume

This long term study demonstrates for the first time that it is possible to propagate embryogenic Vigna trilobata and to subsequently initiate the differentiation of embryos into complete plantlets. Initiation of callus was possible on 2,4-D. Somatic embryos differentiated on modified MS basal nutrient medium with 1.0 mg/l  of 2,4-D and 0.5 mg/l  of Kn. Sustained cell division resulted in globular and heart shape stages of somatic embryos. Transfer of embryos on to a fresh modified MS basal medium with 0.5 mg/l of Kn and 0.5 mg/l of GA3 helped them to attain maturation and germination. However, the propagation of cells, as well as the differentiation of embryos, were inhibited by a continuous application of these growth regulators. For this reason, a long period on medium lacking these growth regulators was necessary before the differentiation of embryos occurred again. The consequences for improving the propagation of embryogenic cultures in Vigna species are discussed. Key words: Pasture  legume, Vigna trilobata, Globular, Heart shape, somatic embryogenesis D.O.I. 10.3329/ptcb.v19i1.4990 Plant Tissue Cult. & Biotech. 19(1): 89-99, 2009 (June)

Dwindling status of Epimedium Elatum (Morren & Decne) and its geographical distribution in Kashmir Himalaya, India

2018 ◽  
pp. 47-52
Keyword(s):  
Medicinal Plant ◽  
Biological Activities ◽  
Conservation Status ◽  
Natural Populations ◽  
Culture Techniques ◽  
Kashmir Himalayas ◽  
Near Future ◽  
First Time ◽  
Tissue Culture Techniques

Epimedium elatum (Morren & Decne) of family Berberidaceace is a rare perennial medicinal plant, endemic to high altitude forests of Northwestern Himalayas in India. Ethnobotanically, it has been used as an ingredient for treatment of bone-joint disorders, impotence and kidney disorders in Kashmir Himalayas. Phytochemically, it is rich in Epimedin ABC and Icariin; all of these have been demonstrated to possess remarkable biological activities like PDE-5 inhibition (treatment of erectile dysfunction), anticancer, antiosteoporosis antioxidant and antiviral properties. The present investigation reports its traditional usage, comprehensive distribution and conservation status from twenty ecogeographical regions in Kashmir Himalayas, India. The species was reported from Gurez valley for the first time. Numerous threats like excessive grazing, deforestration, habitat fragmentation, tourism encroachment, landslides and excessive exploitation have decreased its natural populations in most of the surveyed habitats. Consequently, its existence may become threatened in near future if timely conservation steps are not taken immediately by concerned stakeholders involved in medicinal plant research. Moreover, use of plant tissue culture techniques is recommended for development of its in vitro propagation protocols. Therefore, introduction of this medicinal plant in botanical gardens, protected sites and development of monitoring programmes are needed for its immediate conservation in Northwestern Himalayas, India.

Structure-Reactivity Relationships on Substrates and Inhibitors of the Lysine Deacylase Sirtuin 2 from Schistosoma Mansoni (SmSirt2)

2019 ◽  
Author(s):  
Daria Monaldi ◽  
Dante Rotili ◽  
Julien Lancelot ◽  
Martin Marek ◽  
Nathalie Wössner ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Human Cancer ◽  
Egg Laying ◽  
Human Cancer Cells ◽  
Parasite Survival ◽  
Survival And Reproduction ◽  
Emergence Of Resistance ◽  
First Time ◽  
Parasitic Life Cycle

The only drug for treatment of Schistosomiasis is Praziquantel, and the possible emergence of resistance makes research on novel therapeutic agents necessary. Targeting of Schistosoma mansoni epigenetic enzymes, which regulate the parasitic life cycle, emerged as promising approach. Due to the strong effects of human Sirtuin inhibitors on parasite survival and reproduction, Schistosoma sirtuins were postulated as therapeutic targets. In vitro testing of synthetic substrates of S. mansoni Sirtuin 2 (SmSirt2) and kinetic experiments on a myristoylated peptide demonstrated lysine long chain deacylation as an intrinsic SmSirt2 activity for the first time. Focused in vitro screening of the GSK Kinetobox library and structure-activity relationships (SAR) of identified hits, led to the first SmSirt2 inhibitors with activity in the low micromolar range. Several SmSirt2 inhibitors showed potency against both larval schistosomes (viability) and adult worms (pairing, egg laying) in culture without general toxicity to human cancer cells.<br>

α-Glucosidase Inhibition and Docking Studies of 5-Deoxyflavonols and Dihydroflavonols Isolated from Abutilon pakistanicum

2019 ◽  
Vol 23 (17) ◽  
pp. 1857-1866
Author(s):  
Munawar Hussain ◽  
Zaheer Ahmed ◽  
Shamsun N. Khan ◽  
Syed A. A. Shah ◽  
Rizwana Razi ◽  
...  
Keyword(s):  
Methanolic Extract ◽  
Docking Studies ◽  
Hydroxyl Group ◽  
Coumaric Acid ◽  
In Vitro Activity ◽  
Aerial Parts ◽  
First Time ◽  
Acid Esters ◽  
Phenol Moiety

Three new 5-deoxyflavonoid and dihydroflavonoids 2, 3 and 4 have been isolated from the methanolic extract of Abutioln pakistanicum aerial parts, for which structures were elucidated explicitly by extensive MS- and NMR-experiments. In addition to these, 3,7,4′-trihydroxy-3′-methoxy flavonol (1) is reported for the first time from Abutioln pakistanicum. Compound 2 and 4 are p-coumaric acid esters while compounds 2–4 exhibited α-glucosidase inhibitory activity. Docking studies indicated that the ability of flavonoids 2, 3 and 4 to form multiple hydrogen bonds with catalytically important residues is decisive hence is responsible for the inhibition activity. The docking results signified the observed in-vitro activity quite well which is in accordance with previously obtained conclusion that phenol moiety and hydroxyl group are critical for the inhibition of α-glucosidase enzyme.

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