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 tumefaciens-transient genetic transformation of Habanero pepper (Capsicum chinense Jacq.) leaf explants

2010 ◽  
Vol 13 (4) ◽  
Author(s):  
Guadalupe Fabiola Arcos-Ortega ◽  
Rafael Antonio Chan-Kuuk ◽  
Wilma Aracely González-Kantún ◽  
Ramón Souza-Perera ◽  
Yumi Elena Nakazawa-Ueji ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Leaf Explants ◽  
Capsicum Chinense ◽  
Habanero Pepper

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 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 Comparative Proteomic Analysis on Chloroplast Proteins Provides New Insights Into the Effects of Low Temperature in Sugar Beet

2021 ◽  
Author(s):  
Jiali Long ◽  
Wang Xing ◽  
Yuguang Wang ◽  
Zedong Wu ◽  
Wenjing Li ◽  
...  
Keyword(s):  
Abiotic Stress ◽  
Low Temperature ◽  
Sugar Beet ◽  
Temperature Stress ◽  
Phylogenetic Analyses ◽  
Marker Gene ◽  
Low Temperature Stress ◽  
Chloroplast Envelope ◽  
Pcr Analysis ◽  
Sugar Beets

Abstract Background: Low temperature, which is one of the main environmental factors that limits geographical distribution and sucrose yield, is a common abiotic stress during the growth and development of sugar beet. As a regulatory hub of plant response to abiotic stress, activity in the chloroplasts is related to many molecular and physiological processes, particularly in response to low temperature stress. Results: The contents of chlorophyll (Chl) and malondialdehyde (MDA), relative electrical conductivity (REL), and superoxide dismutase (SOD) activity were measured. The results showed that sugar beet could manage low temperature stress by regulating the levels of Chl, REL and MDA, and the activity of SOD. The physiological responses indicated that sugar beets respond positively to low temperature treatments and are not significantly damaged. Moreover, to determine the precise time to response low temperature in sugar beet, well-known abiotic stresses-responsive transcript factor family, namely DEHYDRATION RESPONSIVE ELEMENT BINDING PROTEIN (DREB), was selected as the marker gene. The results of phylogenetic analyses showed that BvDREBA1 and BvDREBA4 were in the same branch as the cold- and drought-responsive AtDREB gene. In addition, the expression of BvDREBs reached its maximum level at 24 h after low temperature by RNA-Seq and qRT-PCR analysis. Furthermore, the changes in chloroplast proteome after low temperature at 24 h were detected using a label-free technique. A total of 416 differentially expressed proteins were identified. GO enrichment analysis showed that 16 GO terms were significantly enriched, particularly chloroplast stroma, chloroplast envelope, and chloroplast thylakoid membrane. It is notable that the transport of photosynthetic proteins (BvLTD, BvTOC100, and Toc-Tic complex), the formation of starch granules (BvPU1, BvISA3, and BvGWD3) and the scavenging of reactive oxygen species (BvCu/Zn-SOD, BvCAT, BvPrx, and BvTrx) were the pathways used by sugar beets to respond to low temperatures at an early stage.Conclusions: These results provide a preliminarily analysis of how chloroplasts of sugar beet respond to low temperature stress at the translational level and provide a theoretical basis for breeding low temperature resistant varieties of sugar beet.

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 Agrobacterium-Mediated Genetic Transformation of Potato for Abiotic Stress Tolerance

2013 ◽  
Vol 19 (2) ◽  
pp. 37-43
Author(s):  
SN Begam ◽  
KM Nasiruddin ◽  
MS Haque ◽  
S Yasmin
Keyword(s):  
Abiotic Stress ◽  
Genetic Transformation ◽  
Stress Tolerance ◽  
Callus Induction ◽  
Abiotic Stress Tolerance ◽  
Marker Gene ◽  
Leaf Explants ◽  
Nptii Gene ◽  
Ms Medium ◽  
Maximum Percentage

Two potato varieties, Cardinal and Heera, were used to optimize the Agrobacterium-mediated genetic transformation system in potato for abiotic stress tolerance. MS medium supplemented with different concentrations and combinations of NAA and BAP were used to obtain callus induction from leaf explants of potato cv Cardinal and Heera. Maximum percentage of callus induction (95.83%) was observed in MS medium supplemented with 4 mg/L NAA and 2 mg/L BAP. The highest callusing was found in Cardinal and the lowest in Heera. Calluses were inoculated with Agrobacterium tumefaciens strain LBA4404 (pB1121) carrying CIPK sense gene, selectable marker gene nptII gene conferring resistance to kanamycin and the GUS reporter gene. Both the varieties were found GUS positive after inoculation and selection. Cardinal had maximum percentage of survived callus (25%) on selection media. Stable intregration and expression of the transgenes were confirmed by GUS histochemical assay.DOI: http://dx.doi.org/10.3329/pa.v19i2.16919 Progress. Agric. 19(2): 37 - 43, 2008

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