Genetic Transformation for Pod Borer Resistance in Dolichos Bean [Lablab purpureus (L.) Sweet]

2021 ◽  
Author(s):  
J.K. Kshirsagar ◽  
S.V. Sawardekar ◽  
S.G. Bhave ◽  
N.B. Gokhale ◽  
A.L. Narangalkar ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Transformation Frequency ◽  
Vector System ◽  
Pcr Analysis ◽  
Nptii Gene ◽  
Lablab Purpureus ◽  
Cry Genes ◽  
Mendelian Segregation ◽  
Pcr Screening ◽  
Progeny Analysis

Background: Agrobacterium mediated genetic transformation experiments were carried out in Dolichos bean Cv. (Konkan Bhushan) showing better regenerability. Methods: Three cry genes viz. cry1Aabc, cry1Fa1 and cry2Aa were used for the transformation each of which were linked to CaMV35S promoter and nptII gene under control of nos promoter and terminator. A vector system consisting of the disarmed hyper virulent Agrobacterium tumefaciens strain EHA-105 harboring pBinAR or BinBt3 was used. Mature embryo axis with single cotyledon was used as explant. Kanamycin as well as PCR screening was carried out to assess the transformation frequency. Progeny analysis using PCR was also carried out to assess the transgene segregation and stable transformation.Result: Kanamycin concentration of 500 mg/l was found as optimum for selection of a transgenic turning leaf blades albino. Among five methods of colonization used, the method employing mild injury to explant with dipping in Agrobacterium culture for 20 minutes followed by co-cultivation for 48 hours, cefotaxime washing and sowing in soil resulted in maximum survival (74.80%) associated with maximum transformation frequency through PCR analysis (2.13%). Among three cry genes, the gene cry2Aa was found the most effective in transforming Dolichos bean. The progeny analysis of transformants has shown the 3:1 mendelian segregation ratio confirming stable transformation of transgene.

scholarly journals Optimization of genetic transformation method for an indica rice (Oryza sativa L.) variety Ratnagiri-711

2020 ◽  
Vol 80 (02) ◽  
Author(s):  
G. B. Sawant ◽  
S. G. Bhave ◽  
S. V. Sawardekar ◽  
M. M. Burondkar ◽  
N. B. Gokhale ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Shoot Apical Meristem ◽  
Apical Meristem ◽  
Transformation Method ◽  
Transformation Frequency ◽  
Vector System ◽  
Nptii Gene ◽  
Cry Genes ◽  
In Planta ◽  
Germinating Seeds

A study for method optimization of Agrobacterium-mediated genetic transformation for insect resistance was carried out for a rice variety Ratnagiri-711 showing better regenerability. Three different cry genes viz. cry1Aabc, cry1Fa1 and cry2Aa with a vector system consisting of the disarmed hyper virulent Agrobacterium tumefaciens strain EHA-105 harboring pBinAR or BinBt3 were used. The respective genes were linked to CaMV35S promoter and nptII gene under control of nos promoter and terminator. Scutellum-derived callus bits and embryonic shoot apical meristem of germinating seeds were used as target tissues for callus-mediated and in planta transformation, respectively. Kanamycin screening and PCR analysis was employed for confirmation of presence of transgene. Among five methods of colonization and co-cultivation tried with three cry genes, a callus-mediated transformation method consisting of 20 minutes colonization and 3 days co-cultivation with cry2Aa gene recorded highest transformation frequency (13.79%) but minimum survival (5.27%). On the contrary, considerable transformation frequency (6.35%) with highest survival (79.42%) was observed in an in planta method employing mild injury to embryonic shoot apical meristem of germinating seeds followed by injection of Agrobacterium having cry2Aa gene followed by 15 minutes colonization and then directly sowing in pots. Among three cry genes used, the gene cry2Aa was found most effective showing more transformation frequency.

scholarly journals Creation of transgenic sugar beet lines containing synthetic gene cry1C

1970 ◽  
pp. 221-225
Author(s):  
V. V. Kurylo ◽  
E. N. Shysha ◽  
A. I. Yemets
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Sugar Beet ◽  
Genetic Transformation ◽  
Marker Gene ◽  
Leaf Explants ◽  
Plant Genome ◽  
Direct Regeneration ◽  
Pcr Analysis ◽  
Cry Genes ◽  
Sugar Beet Crop

Aim. Insect pest’s impact makes a significant limitation of the sugar beet crop yield. Integration of cry-genes of Bacillus thuringiensis into the plant genome is one of the promising strategies to ensure of plant resistance. The aim of this work was the production of sugar beet lines (based on the MM1/2 line) expressing cry1C genes. Methods. Genetic transformation of sugar beet was performed using the method of co-cultivation of leaf explants with Agrobacterium tumefaciens. Results. Sugar beet line MM1/2 was transformed by Agrobacterium-mediated method of transformation using binary vector pRD400-cry1CST, containing synthetic cry1C gene and selectable marker gene neomycin phosphotransferase II (nptII) conferring resistance to kanamycin. After the optimization protocol of genetic transformation and direct regeneration from leaf discs a transgenic sugarbeet lines were obtained. Conclusions. PCR analysis revealed integration of cry1C into the genome of transgenic lines of B. vulgaris.Keywords: genetic transformation, Agrobacterium tumefaciens, Beta vulgaris, cry-genes.

scholarly journals AGROBACTERIUM-MEDIATED TRANSFORMATION OF TWO TOMATO CULTIVARS (LYCOPERSICON ESCULENTUM MILL.) CV. SANDRA AND ROCKY

2021 ◽  
Vol 52 (3) ◽  
pp. 745-755
Author(s):  
G. H. Danial ◽  
D. A. Ibrahim ◽  
G. Q. Song
Keyword(s):  
Marker Gene ◽  
Kanamycin Resistance ◽  
Transformation Frequency ◽  
Pcr Analysis ◽  
Nptii Gene ◽  
Agrobacterium Mediated Transformation ◽  
Nos Terminator ◽  
Transgenic Tomato Plants ◽  
Tomato Cultivars

An efficient protocol for Agrobacterium-mediated transformation of tomato cultivars Sandra and Rocky was conducted to examine the possibility of producing transgenic tomato plants cultivars harbouring the nptII gene, conferring kanamycin resistance. To achieve this aim, tomato cotyledon explants were transformed using EHA105 Agrobacterium tumefaciens strain harboring the binary vectors pBI121 which contains Gus gene, and neomycin phosphotransferase II (nptII) as selectable marker gene under the control of a CaMV35S promoter and nopaline synthase (nos) Terminator. Transformant detection was carried out in three distinct ways. First antibiotic selection, Kanamycin at a concentration of100 mgl-1 found to be efficient for this purpose. Second histochemical GUS assay revealed the presence of blue colored zones in a number of shoots and leaves for both in vitro and the greenhouse-grown transgenic plants. Third PCR analysis indicated positive result by showing the fragment for nptII gene in tested transformants, while was absent in non-transgenic control (wild type). On the other hand, the results showed that Sandra cultivar was more efficient for regeneration and subsequently transformation frequency than Rocky cultivar, which record 26.66% of transformation frequency compared with 11.57% in Rocky cultivar.

scholarly journals Efficiency of Genetic Transformation via Pollen-Tube Pathway of Jatropha (Jatropha curcas L.) Based on Histochemical and Molecular Analysis

2018 ◽  
Vol 45 (3) ◽  
pp. 316
Author(s):  
Agus Zainudin ◽  
Bambang Sapta Purwoko ◽  
Tri Joko Santoso ◽  
Sintho Wahyuning Ardie ◽  
And Trikoesoemaningtyas
Keyword(s):  
Pollen Tube ◽  
Genetic Transformation ◽  
Molecular Analysis ◽  
Plasmid Dna ◽  
Jatropha Curcas ◽  
Marker Gene ◽  
Pcr Analysis ◽  
Jatropha Curcas L ◽  
Gus Reporter ◽  
Combination Treatments

The genetic transformation via pollen-tube pathway is an alternative method to overcome the constraints imposed by genotype specificity in transformation and regeneration in jatropha (Jatropha curcas L.) tissue culture. Therefore, it is necessary to establish important parameters for efficient genetic transformation of jatropha via pollen-tube pathway. The objective of the research was to study the efficiency of direct transformation of jatropha via pollen-tube pathway based on histochemical and molecular analysis. Solution of purified pCAMBIA1301 DNA plasmid carrying a hptII marker gene and a gus reporter gene with concentration level of 0.05, 0.25, 0.50 µg µl-1 were applied to stigma of flowers at 1, 2, 4, 7, 10 h after pollination. Seedling of IP3A, IP3P and JcUMM18 jatropha’s genotypes derived from 15 combination treatments of plasmid DNA concentration and application time, also wild type was subjected to histochemical and molecular analyses. Based on those analyses, the efficiency of transformation via pollen-tube pathway of three jatropha genotypes ranged from 1.5-16.7%. PCR analysis showed that a number of positive plants were identified by using specific primers hptII and gus, i.e. 1-3 and 3-7 plants of the 15 combined treatments, respectively. It indicated that the transformation efficiency via the pollen-tube pathway varied in each jatropha genotype.<br /><br />Keywords: Jatropha curcas L., pCAMBIA1301, plasmid DNA, stigma-drip<br /><br />

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 Obtaining of transgenic alfalfa (Medicago sativa L.) and peanut (Arachis hypogaea L.) plants resistant to the herbicide Pursuit by Agrobacterium-mediated transformation

1970 ◽  
pp. 243-246
Author(s):  
S. M. Nifantova ◽  
I. K. Komarnytskyi ◽  
M. V. Kuchuk
Keyword(s):  
Gene Selection ◽  
Molecular Size ◽  
Pcr Analysis ◽  
Nptii Gene ◽  
Transformation Protocol ◽  
Genetic Construct ◽  
Agrobacterium Mediated Transformation ◽  
Transgenic Alfalfa ◽  
Arachis Hypogaea L ◽  
Selective Agents

Aim. The production of alfalfa and peanut cultivars with new properties is necessary. The purpose of this work was to develop Agrobacterium-mediated transformation protocol and to construct transgenic alfalfa and peanut plants resistant to herbicide Pursuit Methods. Genetic transformation was carried out using cocultivation of peanut and alfalfa explants with Agrobacterium tumefaciens strain GV3101 carrying genetic construct pCB004 containing mutant ahas/als gene and nptII gene. Selection was held on the solidified callus inducing medium with 50 mkg/l Pursuit. The selected callus clones were put on the regeneration medium with the same selective agents. Obtained regeneration lines were analysed using PCR-analysis. Results. 17 peanut and 14 alfalfa regeneration lines had positive signals after PCR analysis with DNA fragments of required molecular size for ahas/als and nptII genes. Conclusions. Transgenic alfalfa and peanut plants resistant to the herbicide Pursuit were obtained.Keywords: alfalfa, peanut, ahas/als gene, transformation.

scholarly journals Agrobacterium-mediated Genetic Transformation for Local Cultivars of Potato (Solanum tuberosum L.) Using Marker Genes

1970 ◽  
Vol 20 (2) ◽  
pp. 145-155 ◽  
Author(s):  
Rita Sarah Borna ◽  
M. I. Hoque ◽  
R. H. Sarker
Keyword(s):  
Solanum Tuberosum ◽  
Genetic Transformation ◽  
Solanum Tuberosum L ◽  
In Vitro Regeneration ◽  
Direct Regeneration ◽  
Pcr Analysis ◽  
Marker Genes ◽  
Transformation Rate ◽  
Best Responses

Genetic transformation using nodal and internodal segments from three economically important potato (Solanum tuberosum L.) varieties namely, Diamant, Cardinal and Granola was conducted using an Agrobacterium tumefaciens strain LBA4404 harbouring binary plasmid pBI12 containing the GUS and nptII genes. Node and internodal segments were used for direct regeneration as well as regeneration with the intervention of callus. best responses were  obtained for direct regeneration of shoots when the explants were cultured on MS supplemented with 4.0 mg/l BAP +1.0 mg/l IAA, 1.5 mg/l BAP  + 0.5 mg/l IAA and 5.0 mg/l BAP +1.0 mg/l IAA in Diamant, Cardinal  and  Granola, respectively. In Diamant spontaneous in vitro microtuberization was obtained from these proliferated shoots. Further culturing of these in vitro grown green microtubers regenerated a large number of shoots on MS containing 4.0 mg/l BAP +1.0 mg/l IAA. By combining the best treatments, this protocol yielded an average transformation rate of 87% of treared explants. Stable expression of GUS gene was visualized in the various parts of transformed shoots through histochemical assay. Genomic DNA was isolated from transformed shoots and stable integration of the GUS and nptII genes was confirmed by PCR analysis.   Key words:  Potato, in vitro regeneration, transformation   D.O.I. 10.3329/ptcb.v20i2.6894   Plant Tissue Cult. & Biotech. 20(2): 145-155, 2010 (December)

scholarly journals In vitro Regeneration and Agrobacterium-mediated Transformation of Potato (Solanum tuberosum L.) Cultivars Grown in Mexico

2013 ◽  
Vol 22 (2) ◽  
pp. 93-105 ◽  
Author(s):  
Rose Onamu ◽  
Juan P Legaria ◽  
Jaime C Sahagún ◽  
José L Rodríguez ◽  
Joel N Pérez
Keyword(s):  
Solanum Tuberosum ◽  
Solanum Tuberosum L ◽  
In Vitro Regeneration ◽  
Potato Cultivars ◽  
Pcr Analysis ◽  
Nptii Gene ◽  
Histochemical Assay ◽  
Gus Histochemical Assay ◽  
Binary Plasmid

Prior to Agrobacterium-mediated genetic transformation in vitro regeneration protocol was established for three potato cultivars (Alfa, Cambray Rosa Morelos and Atlantic) grown in Mexico using leaf, node and internodal explants. Regeneration protocol was developed with or without the intervention of callus. Two potato cultivars, namely, Cambray Rosa Morelos and Alpha were transformed using Agrobacterium tumefaciens strain LBA4404 harboring binary plasmid pBI121 containing the GUS and nptII genes. GUS histochemical assay and PCR analysis were conducted on rooted shoots grown in media without hormones but supplemented with antibiotics. Transformed shoots tested positive through GUS histochemical assay and integration of nptII gene was confirmed by PCR analysis DOI: http://dx.doi.org/10.3329/ptcb.v22i2.14193 Plant Tissue Cult. & Biotech. 22(2): 93-105, 2012 (December)

Two Functional Mutations in cis of the Ferrochelatase Gene (FECH) Cause Erythropoietic Protoporphyria (EPP).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3544-3544
Author(s):  
Elena Di Pierro ◽  
Valentina Brancaleoni ◽  
Valeria Moriondo ◽  
Maria D. Cappellini
Keyword(s):  
Sequence Analysis ◽  
Promoter Region ◽  
Dna Analysis ◽  
Pcr Analysis ◽  
Intragenic Recombination ◽  
Incomplete Penetrance ◽  
Erythropoietic Protoporphyria ◽  
Mendelian Segregation ◽  
Dominant Disease ◽  
In Cis

Abstract Erythropoietic protoporphyria is an autosomal dominant disease with incomplete penetrance, due to reduced activity of ferrochelatase (FECH), a mitochondrial enzyme that catalyzes the final step of the heme biosynthetic pathway. The clinical phenotype of EPP results from coinheritance of a mutated allele and a wild-type low expressed allele of the FECH gene. To date, more than 100 different mutations have been identified in the FECH gene of EPP patients. Different evidence suggests that an entire haplotype (−251G, IVS1-23T, and IVS3-48C) reduces allele expression. In two Italian EPP patients carring the −250G>C mutation in the promoter region we recently demonstrated the loss of an SP1 binding site causing a strong impairment of promoter activity. In both patients we searched for the GTC haplotype on the other allele. Family segregation studies established that the GTC haplotype was in trans to the −250C mutation. Indeed, asymptomatic parents carried either the three polymorphisms or the point mutation. In both patients only the IVS3-48C polymorphism was in apparent homozygosity since family studies established the absence of Mendelian segregation. To reduce the possibility of interfering polymorphisms in the primer sites, a second set of sequencing primers was designed. Apparent homozygosity was confirmed. We assumed the presence of an intronic deletion on the same allele carrying the −250G>C mutation. In order to establish the size of the deletion we performed a semiquantitative RT-PCR analysis on fresh RNA and XL-PCR analysis on DNA. In both cases the primers were situated in regions outside the heterozygotic polymorphisms. As expected RNA analysis revealed the presence of a normal fragment of 612bp and a shorter fragment of 343bp that was less abundant compared to controls. Sequence analysis on the isolated abnormal fragment showed loss of exons 3 and 4. DNA analysis revealed the presence of two fragments of 21440bp and 15864bp respectively. We performed an internal long PCR to obtain two shorter fragments 9717bp and 4141bp respectively. Sequence analysis on the isolated abnormal fragment showed a 5576bp deletion defined by two short direct repeats of about 40bp. The deleted region includes both a first repeat and interposed sequence. These results showed the presence of two functional mutations on the same allele. Deletion contributed to create a null allele preventing formation of the correct mRNA that is already strongly impaired by the −250G>C mutation. The deletion is caused by slipped strand mispairing or more probably by unequal intragenic recombination. These two mutations in the promoter region and intron 3 respectively, are located in the same regions as the −251G and ivs3-48C polymorphisms, strongly suggesting a functional relationship between these two regions.

Evaluation of transgenic canola plants under field conditions

Genome ◽  
1992 ◽  
Vol 35 (1) ◽  
pp. 58-63 ◽  
Author(s):  
M. Arnoldo ◽  
C. L. Baszczynski ◽  
G. Bellemare ◽  
G. Brown ◽  
J. Carlson ◽  
...  
Keyword(s):  
Kanamycin Resistance ◽  
Genetically Engineered ◽  
Field Conditions ◽  
Enzyme Assays ◽  
Nptii Gene ◽  
Neomycin Phosphotransferase ◽  
Agrobacterium Mediated Transformation ◽  
Transgenic Canola ◽  
Progeny Analysis ◽  
Transformation Vectors

Eleven independent transgenic canola (Brassica napus ssp. oleifera L. cv. Westar and Regent) lines were evaluated in the field. The plants carried a neomycin phosphotransferase (NPTII) gene for kanamycin resistance that was introduced via Agrobacterium-mediated transformation. NPTII enzyme assays, Southern blot hybridizations and progeny analysis, confirmed the stable, heritable integration and expression of the introduced NPTII gene. A number of agronomic characteristics evaluated under field conditions, including maturity, yield, and oil and protein content, were all statistically comparable between the transformed and nontransformed plants. These results indicate that canola can be genetically engineered successfully, and that the Agrobacterium-based transformation system employed does not induce any adverse effects on the intrinsic agronomic and qualitative traits critical to the agricultural industry.Key words: transgenic field trial, canola, Agrobacterium-mediated transformation, vectors.

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