scholarly journals Agrobacterium-Mediated Stable Transformation of Medicinal Plant Andrographis paniculata Callus Expressing β-glucuronidase (GUS) Gene

2015 ◽  
Vol 18 (2) ◽  
pp. 92
Author(s):  
Erly Marwani ◽  
Agustina Tangapo ◽  
Fenny Martha Dwivany
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Gus Expression ◽  
Selective Medium ◽  
Stable Transformation ◽  
Andrographis Paniculata ◽  
Pcr Analysis ◽  
Induction Medium ◽  
Gus Gene ◽  
Glucuronidase Activity ◽  
Leaf Disks

This study was carried out to establish a stable genetic transformation in callus culture of Andrographispaniculata mediated by Agrobacterium tumefaciens. The leaf disks of A. paniculata were infected with A. tumefaciensLBA4404 carrying a binary vector pCAMBIA1304 that contain β-glucuronidase (GUS) and hygromycinphosphotransferase (hpt) genes. The infection was conducted by dipping method for one hour, followed byco-cultivation in the dark for three days. To examine transient GUS expression, the co-cultivated leaf disks wereassayed for β-glucuronidase activity and to obtain stable transformed callus, the co-cultivated leaf disks wereselected on the callus induction medium which contain 20 mg/l hygromycin for selection. The transformedcallus was periodically subcultured every three weeks into the fresh selection medium over the 15 weeksperiod. To test a stable transformation, the callus was subjected to PCR analysis for GUS gene detection. Theresults indicated that the co-cultivated leaf disks expressed GUS activity and proliferated to produce callus onthe selective medium. Analysis of PCR on the transformed callus indicated the presence 976 bp fragment thatconfi rmed the presence of β-glucuronidase gene. These fi ndings imply that the β-glucuronidase was stably integrated into A. paniculata callus culture.Keywords: Andrographis paniculata, Agrobacterium tumefaciens, andrographollide, transformed callus,β-glucuronidase gene.

scholarly journals Agrobacterium-mediated Genetic Transformation of Two Varieties of Jute (Corchorus capsularis L.)

1970 ◽  
Vol 18 (1) ◽  
pp. 7-16 ◽  
Author(s):  
Rakha Hari Sarker ◽  
G.M. Al-Amin ◽  
Fathi Hassan ◽  
M.I. Hoque
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Plant Tissue ◽  
Genomic Dna ◽  
Mature Embryo ◽  
Gus Expression ◽  
Pcr Analysis ◽  
Gus Gene ◽  
Corchorus Capsularis ◽  
Selection Of

Transformation experiments were carried out using different explants of two varieties of white jute (Corchorus capsularis L.), namely, CVL-1 and CVE-3 with Agrobacterium tumefaciens strain (LBA4404/pBI121) containing the GUS and nptII genes. Maximum transformation ability was obtained from petiole-attached cotyledons and mature embryo explants. Kanamycin at a concentration of 200 mg/l was found optimum for selection of transformed shoots developed frommature embryos. Histochemical assay revealed the stable expression of the GUS gene within the various tissues of transformed plantlets. Stable integration of GUS and nptII genes were confirmed by PCR analysis of genomic DNA isolated from these transformed shoots. Key words: Jute, Transformation, GUS expression, PCR analysis D.O.I. 10.3329/ptcb.v18i1.3245 Plant Tissue Cult. & Biotech. 18(1): 7-16, 2008 (June)

scholarly journals Application of sonication-assisted Agrobacterium-mediated transformation in Chenopodium rubrum L

2011 ◽  
Vol 49 (No. 6) ◽  
pp. 255-260 ◽  
Author(s):  
J.I. Flores Solís ◽  
P. Mlejnek ◽  
K. Studená ◽  
S. Procházka
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Transient Expression ◽  
Chenopodium Rubrum ◽  
Limiting Factors ◽  
Gus Gene ◽  
Efficient Tool ◽  
Factors Affecting ◽  
Agrobacterium Mediated Transformation ◽  
Glucuronidase Activity ◽  
Gene Coding

Chenopodium rubrum belongs to the plant species in which standard Agrobacterium-mediated transformation procedures remain inefficient. We demonstrate that the employment of sonication-assisted Agrobacterium-mediated transformation (SAAT) effectively enhanced transient expression of GUS gene coding for b-glucuronidase in Chenopodium rubrum. Further the results indicated that the age of seedlings is one of the limiting factors affecting the potency of Agrobacterium tumefaciens infection. Histochemical detection of b-glucuronidase activity revealed that two-days-old seedlings were much more susceptible to infection than ten-days-old ones. According to our results SAAT technology could provide an efficient tool for obtaining stable transformants when applied to two-days-old seedlings of Chenopodium rubrum.

scholarly journals EXPRESSION OF REPORTER GENES IN TRANSFORMED INDICA RICE THROUGH Agrobacterium MEDIATED METHOD

2007 ◽  
Vol 20 (1) ◽  
pp. 01-08
Author(s):  
Shamsul H. Prodhan ◽  
A. Komamine
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Oryza Sativa L ◽  
Indica Rice ◽  
Kanamycin Resistance ◽  
Pcr Analysis ◽  
Gus Gene ◽  
Immature Inflorescence ◽  
Seed Fertility ◽  
Selection Medium ◽  
Mature Seeds

Genetic transformation of rice (Oryza sativa L.) mediated by Agrobacterium tumefaciens has been confirmed for japonica varieties and extended to include more recalcitrant indica varieties. Scutellum-derived calli from mature seeds of Kasalath and BR-5 were used. The Agrobacterium tumefaciens strain, EHA101, harboring the binary vector pIG121Hm/Km/GUS was used for transformation. The vector contains b-glucuronidase (GUS) gene as a reporter gene and hygromycin resistance (HPT) as well as kanamycin resistance gene (NPTII) as selection genes in the T-DNA region. After co-cultivation with the bacteria, calli were inoculated on selection medium in which hygromycin concentration was 50 mg/l for Kasalath and 20 mg/l for BR-5. Carbenicillin (500 mg/l) was used for removal of Agrobacterium after co-cultivation. Inclusion of acetosyringone 50–100 mM in the Agrobacterium suspension in co-culture medium increased the frequency of transformation. Frequency of transformed calli (hygromycin resistant cells) was 82% in Kasalath and 6% in BR-5. Regeneration efficiency from transformed calli in Kasalath was about 63% and in BR-5 was about 34%. Most of the transgenic plants were morphologically normal but seed fertility was lower than the control. In transformed calli, roots and immature inflorescence showed positive response in GUS assay. Presence of GUS, HPT and NPTII genes was confirmed by PCR analysis and PCR Southern blot analysis. Expression of GUS gene was 100% in T1 progeny of Kasalath, whereas that of HPT gene was 51%. BR-5 could not be tested because of low seed fertility of T0 plants. In T1 plants, seed fertility of transformed Kasalath was 79% which is lower than that of the respective non-transformants.DOI: http://dx.doi.org/10.3329/bjpbg.v20i1.10633

scholarly journals Optimization of Agrobacterium Mediated Genetic Transformation in Paspalum scrobiculatum L. (Kodo Millet)

Agronomy ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1104
Author(s):  
Ritika Bhatt ◽  
Prem Prakash Asopa ◽  
Rohit Jain ◽  
Aditi Kothari-Chajer ◽  
SL Kothari ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Transgenic Plants ◽  
Genetic Transformation ◽  
Transformation Efficiency ◽  
Normal Growth ◽  
Induction Medium ◽  
Ms Medium ◽  
Gus Gene ◽  
Cultivation Medium ◽  
Kodo Millet

An efficient and reproducible protocol for Agrobacterium tumefaciens mediated genetic transformation was developed for kodo millet (Paspalum scrobiculatum L.) by optimizing various parameters. Agrobacterium strains EHA 105 and LBA 4404 harboring plasmids pCNL 56 and pCAMBIA 2300, respectively, provided the highest transformation efficiency. Addition of acetosyringone (AS) in infection medium (200 µM EHA 105, 250 µM–LBA 4404) and co-cultivation medium (50 µM) increased the transformation efficiency. Transient and stable expression of gus gene was confirmed with histochemical assay of infected embryos and leaves of transformed plants, respectively. The best GUS response was obtained by pretreatment of callus with an antinecrotic mixture (10 mg/L Cys + 5 mg/L Ag + 2.5 mg/L As) at infection time of 20 min followed by co-cultivation for 3 days (EHA 105) and 5 days (LBA 4404) in dark. Regenerated transgenic plants were obtained after 8 to 10 weeks of selection on callus induction medium (NAA 0.5 mg/L, BAP 1 mg/L) containing 50 mg/L Kan + 250 mg/L Cef and were rooted for 2 weeks on MS medium containing PAA (1 mg/L) and phytagel. The plantlets established in greenhouse showed normal growth. Therefore, the protocol developed in the present study can be used for development of improved varieties of kodo millet.

scholarly journals TRANSIENT GENE EXPRESSION IN SPINACH CALLUS TRANSFORMED WITH AGROBACTERIUM TUMEFACIENS

HortScience ◽  
1992 ◽  
Vol 27 (6) ◽  
pp. 621c-621 ◽  
Author(s):  
J.M. Al-Khayri ◽  
F.H. Huang ◽  
T.E. Morelock ◽  
H.T. Zhang
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Spinacia Oleracea ◽  
Transient Gene Expression ◽  
Kanamycin Resistance ◽  
Ms Medium ◽  
Gus Gene ◽  
Selection Medium ◽  
High Level ◽  
Overnight Culture ◽  
Leaf Disks

The objective of this study was to determine the efficacy of Agrobacterium tumefaciens in transforming spinach (Spinacia oleracea L.) callus. Callus was induced from leaf disks of `Baker' on Murashige and Skoog (MS) medium supplemented with 2 mg L-1 kinetin and 0.5 mg L-1 2,4-D. Callus was cut into 2-mm pieces, and 0.5 g of callus was placed in each 250-ml flask which contained 20 ml of MS liquid medium. The suspension cultures were inoculated with 100 μl of an overnight culture of A. tumefaciens harboring pMON 9749 (provided by S. Rogers, Monsanto Co., St. Louis), a plasmid cointegrated with kanamycin resistance and β -glucuronidase (GUS) genes. After coculturing for 2 days at 22C with shaking at 100 rpm, the medium was replaced with selection medium containing (in μg/ml) 75 kanamycin, 100 cefotaxime, and 200 carbenicillin and maintained for 3 weeks. Transient expression of GUS gene in transformed cells was detected with X-glu assay. This method resulted in a high level of transformation and provides the first report of transformation in spinach. This study was funded by a grant (92-B-32) from the Arkansas Science & Technology Authority.

scholarly journals “Floral-dip” transformation of Amaranthus caudatus L. and hybrids A. caudatus × A. paniculatus L.

Biologija ◽  
2019 ◽  
Vol 64 (4) ◽  
Author(s):  
Olha Yaroshko ◽  
Maksym Vasylenko ◽  
Alena Gajdošová ◽  
Bogdan Morgun ◽  
Olesia Khrystan ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Lethal Dose ◽  
Pcr Analysis ◽  
Gus Gene ◽  
Gene Vector ◽  
Amaranthus Caudatus ◽  
Herbicide Resistant ◽  
Floral Dip ◽  
Transgenic Seeds ◽  
Positive Results

After “floral-dip” transformation of Amaranth plants with Agrobacterium tumefaciens strain GV3101 carrying pCBV19 gene vector that contained bar and gus genes, transgenic seeds were obtained. The functioning of the tran+sferred genes in Amaranthus tissues was confirmed with herbicide selection (PPT herbicide – phospinotricin) and gus gene activity. Positive results were obtained for cultivars “Karmin” and “Kremoviy rannii”. The percentage of GUS positive samples was 1% (for “Karmin”), 2.2% (for “Kremoviy rannii”) from the total initial quantity of plants that was prior to selection with the herbicide. The seeds of six amaranth cultivars were received after treatment with A. tumefaciens by the method “floral dip”. The lowest lethal dose of herbicide PPT was established – 40 mg/l. After spraying with herbicide, resistant plants were obtained for cultivars: “Kremoviy rannii” (21%) and “Karmin” (20%). After conduction of PCR analysis, positive results were obtained for four cultivars. The percentage of bar positive plants was 0.3% (“Helios”); 0.26% (“Sterkch”); 0.06% (“Kremoviy rannii”); 0.3% (“Rushnichok”) from total initial quantity of plants.

scholarly journals Transformasi dan Ekspresi Transien Gen Pelapor Gusa pada Andrographis paniculata (Burm.F.) Wallich Ex Ness (Transformation and Expression of Reporter Gene Gusa on Andrographis paniculata (Burm.F.) Wallich Ex Ness)

2012 ◽  
Vol 2 (1) ◽  
Author(s):  
Agustina Tangapo
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Reporter Gene ◽  
Selectable Marker ◽  
Binary Vector ◽  
Leaf Explants ◽  
Selective Medium ◽  
Andrographis Paniculata ◽  
Transformation Procedure ◽  
Gusa Gene ◽  
Selection Agent

AbstrakAndrographis paniculata diketahui mengandung senyawa andrografolid, yaitu suatu metabolit sekunder yang memberikan efek farmakologi berupa hepatoprotektif, antiviral dan antikanker. Dalam penelitian ini dilaporkan studi awal prosedur transformasi genetik A. paniculata dengan perantara Agrobacterium tumefaciens. Eksplan daun A. paniculata diinkubasi dengan Ag. tumefaciens strain LBA4404 yang mengandung vektor ganda pCAMBIA1304 dengan gen hpt sebagai gen penanda untuk resistensi higromisin dan gen gusA sebagai gen pelapor. Setelah kokultivasi, eksplan daun dikultur pada medium seleksi yang mengandung higromisin 20 mg L-1 dan sefotaksim 400 mg L-1. Hasil uji histokimia GUS pada potongan daun setelah tiga hari kokultivasi menunjukkan ekspresi transien GUS mencapai 18,83%. Sebanyak 64,44% jaringan A. paniculata yang telah berhasil ditransformasi menunjukkan regenerasi sel dengan menghasilkan kultur kalus transforman pada medium yang mengandung 20 mg/L higromisin.Kata kunci: transformasi genetik, Agrobacterium tumefaciens, Andrographis paniculata, asai GUS.AbstractAndrographis paniculata is known to contain andrographolide, a secondary metabolite which shows pharmacology effects such as hepatoprotective, antiviral and anticancer. We established an Agrobacterium tumefaciens-mediated transformation procedure for A. paniculata. Leaf explants of A. paniculata were incubated with Ag. tumefaciens strain LBA4404 containing a binary vector pCAMBIA1304 with the hpt gene as a selectable marker for hygromycin resistance and an gusA gene as a reporter gene. Following co-cultivation, leaf explants were cultured on selective medium containing 20 mg L-1 hygromycin and 400 mg L-1 cefotaxime. GUS assays showed that only 18.83 % transformation frequency was obtained in leaf disk tissues after 3 days co-cultivation. As much as 64.44 % of the transformed tissue on MS medium containing selection agent 20 mg/L hygromycin showed cell regeneration to produce calluses.Keywords: genetic transformation, Agrobacterium tumefaciens, Andrographis paniculata, GUS assay.

scholarly journals Transformasi Genetik Tembakau dengan Gen Cold Shock Protein melalui Perantara Agrobacterium tumefaciens

2016 ◽  
Vol 9 (2) ◽  
pp. 58
Author(s):  
Seagames Waluyo ◽  
Sustiprijatno Sustiprijatno ◽  
Suharsono Suharsono
Keyword(s):  
Abiotic Stress ◽  
Agrobacterium Tumefaciens ◽  
Cold Shock ◽  
Stress Condition ◽  
Selective Medium ◽  
Cold Shock Protein ◽  
Pcr Analysis ◽  
Ubiquitin Promoter ◽  
Nos Terminator ◽  
Abiotic Stress Condition

<p>Cold<br />shock protein (Csp) essential for organisms to survive in<br />abiotic stress condition. CspB gene has been fused to<br />ubiquitin promoter in the T-DNA region of pCambia 1300int,<br />and introduced into Agrobacterium tumefaciens LBA4404.<br />This research had an objective to transform genetically<br />Nicotiana tabacum cv. Samsun by CspB gene under the<br />control of Ubiquitin promoter and NOS terminator mediated<br />by A. tumefaciens. Leaf discs were co-cultivated with A.<br />tumefaciens LBA 4404. Based on the number of hygromycinresistant<br />calli, the efficiency of transformation was 57.5%. In<br />the selective medium containing 50 μg/l hygromycin, the<br />efficiency of regeneration of transgenic shoots was 82.6%.<br />Based on PCR analysis using primers corresponding to<br />ubiquitin promoter and CspB gene, 18 putative tobacco<br />transgenic containing CspB gene under the control of<br />ubiquitin promoter.</p>

scholarly journals Agrobacterium tumefaciens-mediated Transformation of Embryogenic Callus and Somatic Embryos of the Banana cv “Ambon Lumut” (Musa acuminata)

2017 ◽  
Vol 2 (6) ◽  
pp. 599 ◽  
Author(s):  
Tifa R. Kusumastuti ◽  
Rizkita R. Esyantia ◽  
Fenny M. Dwivany
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Embryogenic Callus ◽  
Somatic Embryos ◽  
Crop Improvement ◽  
Pcr Analysis ◽  
Abiotic Factor ◽  
Gfp Gene ◽  
Culture Transformation ◽  
Biotechnological Approach

Banana is one of the major fruit crops, though its conventional breeding has limitations, such as sterility and high polyploidy  levels.  Biotechnological  approach  using genetic  transformation  crop for improvement  offers  an alternative  solution.  In  this  study  a  protocol  was developed  for  establishing genetic  transformation  from embryogenic callus and somatic embryos of the banana cv Ambon Lumut . Embryogenic callus was obtained in ID4 medium (MS-based medium) supplemented with 1 mg L-1 IAA, 4 mg L-1 2,4D, and 0.03 g L-1 active charcoal. Embryogenic callus was transferred into liquid mediu m to establish somatic embryos. Embryogenic callus and somatic embryos were used for Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain A GL1, containing pART-TEST7 p lasmid with gfp gene as a reporter and CaM V35S as a promoter, was used for transformations. The embryogenic callus and somatic embryos were transformed using heat-shock method followed by centrifugation  (2000 rpm) and co-cult ivation in liquid medium containing acetosyringone (100 M) for 3 days. Results of the GFP analysis showed transient expression from gfp gene reporter in transformed embryogenic callus and somatic embryos. Transformation efficiency in somatic embryos (85,9%) was higher than  that in embryogenic callus (32.09%). PCR analysis using CaMV primer showed bands that compatible with CaMV35S promoter at 507 bp. This is a report showing establisment of embryogenic callus and somatic embryo culture transformation by using A. tumefaciens-mediated transformation protocol of the local banana cv Ambon Lumut. This study proved  the huge potential for genetic transformation of banana cv Ambon Lumut for crop improvement, such as pest or disease  resistance and abiotic factor stress tolerance. Keywords: banana; embryogenic callus; somatic embryos.

GUS Gene Transformation in Rice (Oryza sativa L.) Variety BRRI Dhan-30 Mediated by Agrobacterium tumefaciens

2008 ◽  
Vol 7 (3) ◽  
pp. 530-536 ◽  
Author(s):  
M. Asaduzzama ◽  
M.A. Bari ◽  
M. Rahman ◽  
M. Minami ◽  
K. Matsushima ◽  
...  
Keyword(s):  
Oryza Sativa ◽  
Agrobacterium Tumefaciens ◽  
Oryza Sativa L ◽  
Gene Transformation ◽  
Gus Gene
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