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 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 Local Cultivars of Chickpea (Cicer arietinum L.)

2012 ◽  
Vol 22 (1) ◽  
pp. 41-50
Author(s):  
Ripa Akter Sharmin ◽  
Jasmin Akter ◽  
R. H. Sarker ◽  
M. I. Hoque
Keyword(s):  
Genetic Transformation ◽  
Cicer Arietinum ◽  
Genomic Dna ◽  
Nptii Gene ◽  
Gus Gene ◽  
Transformation Protocol ◽  
Cicer Arietinum L ◽  
Gus Histochemical Assay ◽  
Binary Plasmid ◽  
Selection Of

Agrobacterium-mediated genetic transformation protocol was established for two chickpea (Cicer arietinum L.) varieties, namely Barichhola-4 (Bch-4) and Barichhola-5 (Bch-5). Transformation ability of different explants such as decapitated embryo with single cotyledon disc (DEC), decapitated embryo (DE) and slice embryo decapitated at shoot end with single cotyledon disc (SEC) were tested using Agrobacterium tumefaciens strain LBA4404 harbouring binary plasmid pBI121, containing the GUS and nptII genes. Maximum transformation ability was exhibited by explants of decapitated embryo (DE) from Barichhola-5 (Bch-5). The optimum regeneration from the transformed tissue was achieved on MS  supplemented with 0.5 mg/l BAP, 0.5 mg/l Kn and 0.2 mg/l NAA along with double the amount of CaCl2 and KNO3. Selection of the transformed shoots was carried out by gradually increasing the concentration of kanamycin to 150 mg/l. Stable expression of the GUS gene was detected in various parts of the transformed shoots through GUS histochemical assay. Stable integration of nptII gene within the genomic DNA from these transformed shoots was confirmed through PCR analysis.DOI: http://dx.doi.org/10.3329/ptcb.v22i1.11258 Plant Tissue Cult. & Biotech. 22(1): 41-50, 2012  (June)

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.

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.

Genetic Transformation of Musa acuminata cv. Vaibalhla (AAA) using Agrobacterium tumefaciens

2017 ◽  
Vol 5 (2) ◽  
pp. 120-131
Author(s):  
Lalremsiami Hrahsel ◽  
◽  
Adreeja Basu ◽  
Lingaraj Sahoo ◽  
Robert Thangjam ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Basal Medium ◽  
Male Flower ◽  
Musa Acuminata ◽  
Vacuum Infiltration ◽  
Bacterial Suspension ◽  
Immature Male ◽  
Selection Of ◽  
Selection Of Transformants

Agrobacterium-mediated genetic transformation of Musa acuminata cv. Vaibalhla (AAA) was performed using Agrobacterium tumefaciens strain EHA105 harboring pCAMBIA2301AtNHX1 plasmid. For the transformation efficiency assay, the un-and pre-cultured immature male flower explants were subjected to various methods of injury such as hypodermal needle injury, with and without sonication and vacuum infiltration. The explants were then inoculated in bacterial suspension by occasional shaking for 30 minutes. After inoculation, explants were co-cultivated for 3 days in dark condition at 22° C in a liquid MS basal medium supplemented with 100 µM acetosyringone. For selection of transformants, the treated explants were cultured on Murashige and Skoog (MS) medium supplemented with BAP (2 mg/L), NAA (0.5 mg/L) and ascorbic acid (75 mg/L) along with antibiotics kanamycin (100 mg/L), cefotaxime (300 mg/L) and augmentin (300 mg/L).The putative transformation was analyzed using histochemical GUS assay. 14 and 21 days pre-cultured male flower explants subjected to 4-5 needle point injury resulted in 93.33% and 100% putative transformation respectively. 30s sonication combined with 5 minutes of vacuum infiltration for 7, 14 and 21 days pre-cultured immature male flower explants gave 73.33%, 66.66% and 71.42% putative transformation respectively.

scholarly journals EFFECT OF VIR GENE INDUCERS ON GENETIC TRANSFORMATION FREQUENCIES OF SWEET POTATO AND GARDEN EGG PLANT

HortScience ◽  
1992 ◽  
Vol 27 (6) ◽  
pp. 660d-660
Author(s):  
Essie T. Blay ◽  
Anant Porobo-Dessai ◽  
C. S. Prakash
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Sweet Potato ◽  
Galacturonic Acid ◽  
Ipomoea Batatas ◽  
Gus Expression ◽  
Ms Medium ◽  
Binary Vectors ◽  
Egg Plant ◽  
Very High

Explants of sweet potato (Ipomoea batatas) and garden egg plant (Solanum integrifolium) were cocultivated with disarmed strains of Agrobacterium tumefaciens containing binary vectors with gusA, gusA-nptll fusion or gusA-intron genes. We examined whether the addition of vir gene inducers during cocultivation would improve the transformation in both crops. Acetosyringone and galacturonic acid were tested individually and in combination. A very high GUS expression was detected histochemically in both plant species. The frequency and extent of transformation varied with the type of explant, petioles being the most responsive. The presence of the vir inducing substances in the medium influenced the percent explant area transformed but did not appreciably affect the frequency of transformation. The selective proliferation of the transformed tissue and organogenesis was achieved by the culture of explants on MS medium supplemented with antibiotics.

scholarly journals Agrobacterium-mediated Genetic Transformation of Lentil (Lens culinaris Medik.) with Chitinase Gene followed by In vitro Flower and Pod Formation

2019 ◽  
Vol 29 (1) ◽  
pp. 99-109
Author(s):  
Subroto K Das ◽  
Kishwar Jahan Shethi ◽  
MI Hoque ◽  
RH Sarker
Keyword(s):  
Genetic Transformation ◽  
Lens Culinaris ◽  
Target Gene ◽  
Gene Selection ◽  
Chitinase Gene ◽  
Pcr Analysis ◽  
Agrobacterium Strain ◽  
Half Strength ◽  
Selection Of

To investigate the integration of chitinase gene in lentil (Lens culinaris Medik.) namely, BARI masur-4 (BM-4), BARI masur-5 (BM-5) and BARI masur-6 (BM-6) through Agrobacterium-mediated genetic transformation was performed using Agrobacterium strain EHA 105 harboring bar (resistant to phosphinotrycin) and chitinase (gene of interest) gene. Selection of transformed shoots was carried out by gradually increasing the concentration of phosphinotrycin (PPT) up to 2.0 mg/l. Transgenic lentil shoots were produced with an overall frequency of 0.36 in case of BM-4 and BM-6 and 0.34 in case of BM-5, respectively. Most of the selected shoots developed in vitro flowers and pods following their sub-culture on half strength of MS supplemented with 20 mg/l IBA, 0.5 mg/l NAA with 50 mg/l ticarcillin. Seedlings germinated from the seeds were successfully transferred to soil for the development of further progeny. Stable integration of target gene was confirmed through PCR analysis. Plant Tissue Cult. & Biotech. 29(1): 99-109, 2019 (June)

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 OPTIMIZATION OF FACTORS AFFECTING THE Agrobacterium tumefaciens- MEDIATED TRANSFORMATION OF Eucalyptus saligna

2018 ◽  
Vol 41 (3) ◽  
Author(s):  
Yohana de Oliveira-Cauduro ◽  
Lais Gomes Adamuchio ◽  
João Carlos Bespalhok Filho ◽  
Isabel Rodrigues Gerhardt ◽  
Juliana Degenhardt-Goldbach ◽  
...  
Keyword(s):  
Gene Expression ◽  
Agrobacterium Tumefaciens ◽  
Culture Medium ◽  
Culture Media ◽  
Gus Gene ◽  
Eucalyptus Saligna ◽  
Factors Affecting ◽  
Gus Gene Expression ◽  
Positive Effect ◽  
Selection Of

ABSTRACT This study aimed to evaluate the effect of factors that may affect the genetic transformation of cotiledonary explants of Eucalyptus saligna mediated by EHA105 strain of Agrobacterium tumefaciens. The vector pBI121 carrying gus gene under control of 35S CaMV promoter was used. The effect of the following factors was evaluated: explant pre-culture, use of different antibiotics and presence of acetosyringone (AS) in co-culture media. An antioxidant solution was also used during excision, containing ascorbic acid (250mg.L-1), citric acid (25mg.L-1) and PVP-40 (1g.L-1). Pre-culture of the explants before the co-culture with bacteria was done over a 4-day period in MS culture medium supplemented with 4.4µM BAP and 2.7ìM NAA. After theco-culture period, three concentrations of kanamycin (12.5;25 and 50mg.L-1) combined with 300mg.L-1 Augmentin® in the culture medium were tested The influence of the antibiotic was also evaluated by keeping the explants in a medium containing 50mg.L-1 Km and 300mg.L-1 Augmentin® or 500mg.L-1 cefotaxime. It was concluded that Augmentin® stimulates organogenesis, that a Km concentration of 12.5mg.L-1 allows selection of explants transformed with gus gene and, finally, the addition of AS (50ìM) to the liquid and solid co-culture media has a positive effect on gus gene expression. Moreover, the use of an antioxidant solution during cotyledon excision is dispensable and the pre-culture of the explants has no effect on bud regeneration or gus gene expression. A transformation efficiency of 1.5% was reached.

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