scholarly journals LaCl3 Treatment Improves Agrobacterium-mediated Immature Embryo Genetic Transformation Frequency of Maize

2021 ◽  
Author(s):  
Shengnan Liu ◽  
Yunlu Shi ◽  
Fang Liu ◽  
Yan Guo ◽  
minhui Lu
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Molecular Breeding ◽  
Channel Blocker ◽  
Immature Embryo ◽  
Transformation Frequency ◽  
Immature Embryos ◽  
Infection Frequency ◽  
Novel Method ◽  
Regenerated Plantlets

Abstract Agrobacterium-mediated genetic transformation of immature embryos is important for gene-function studies and molecular breeding of maize. However, the relatively low genetic transformation frequency remains a bottleneck for applicability of this method, especially on commercial scale. We report that pretreatment of immature embryos with LaCl3 (a Ca2+ channel blocker) improves the infection frequency of Agrobacterium tumefaciens, increases the proportion of positive calluses, yields more positive regenerated plantlets, and increases the transformation frequency from 8.40% to 17.60% for maize. This optimization is a novel method for improving the frequency of plant genetic transformations mediated by Agrobacterium tumefaciens.

scholarly journals LaCl3 Treatment Improves the Agrobacterium-mediated Immature Embryo Genetic Transformation Efficiency in Maize

2021 ◽  
Author(s):  
Shengnan Liu ◽  
Yunlu Shi ◽  
Yan Guo ◽  
minhui Lu
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Molecular Breeding ◽  
Channel Blocker ◽  
Transformation Efficiency ◽  
Immature Embryo ◽  
Scale Production ◽  
Commercial Scale ◽  
Regenerated Plantlets ◽  
Auxiliary Role

Abstract Agrobacterium mediated genetic transformation of immature embryo plays an important auxiliary role in the study of gene function and molecular breeding in maize. However, the relatively low genetic transformation efficiency is still the bottleneck of the application of this method, especially in commercial scale production application. In this study, we found that pretreatment of immature embryos with LaCl 3 , a Ca 2+ channel blocker, could improve the infection efficiency of Agrobacterium tumefaciens , increase the proportion of resistant calluses, obtain more positive regenerated plantlets, and finally improve the transformation efficiency in maize. This optimization provides a new direction for improving the efficiency of plant genetic transformation mediated by Agrobacterium tumefaciens .

scholarly journals Optimasi Konsentrasi Asetosiringon dan Higromisin dalam Transformasi Genetik Padi Fatmawati dengan Perantaraan Agrobacterium tumefaciens

2019 ◽  
Vol 46 (3) ◽  
pp. 223-230
Author(s):  
Atmitri Sisharmini ◽  
Bambang Sapta Purwoko ◽  
Nurul Khumaida ◽  
Dan Kurniawan Rudi Trijatmiko
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Transformation Efficiency ◽  
Immature Embryo ◽  
Cultivation Medium ◽  
Transformation Protocol ◽  
Randomized Design ◽  
Transgenic Lines ◽  
Completely Randomized Design ◽  
Design Factors

Protocols for genetic transformation of rice have been widely developed, however the protocols are not universal and inapplicable for all types of rice plants directly. Transformation protocol on rice cv. Fatmawati needs to be developed to generate transgenic lines. The present research was carried out to optimize genetic transformation protocol in rice cv. Fatmawati mediated by Agrobacterium tumefaciens harboring pCambia1301 construct using immature embryo as an explant. The experiment was arranged in a completely randomized design. Factors influencing efficiency of transformation, i.e., sensitivity of callus to hygromycin antibiotic, acetosyringone concentration used in cultivation medium, hygromycin concentration for transformant selection were optimized. The results showed that genetic transformation of rice cv. Fatmawati mediated by A. tumefaciens using immature embryos have been successfully carried out with several parameters. Addition of 100 µM acetosyringone in co-cultivation medium and 30 mg L-1 hygromycin for transformant callus selection were optimal for genetic transformation of rice cv. Fatmawati mediated by A. tumefaciens. Transformation efficiency was found to be 7.84% based on the lines carrying the hpt gene. This result would be a valuable reference in genetic transformation of rice cv. Fatmawati using target genes.Keywords: immature embryo, Oryza sativa, pCambia1301, transformation efficiency

Genetic transformation of Ginkgo biloba by Agrobacterium tumefaciens

2000 ◽  
Vol 108 (4) ◽  
pp. 413-419
Author(s):  
Patricia Dupré ◽  
Jerôme Lacoux ◽  
Godfrey Neutelings ◽  
Dominique Mattar-Laurain ◽  
Marc-André Fliniaux ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Ginkgo Biloba

scholarly journals Optimizing Transformation Frequency of Cryptococcus neoformans and Cryptococcus gattii Using Agrobacterium tumefaciens

2021 ◽  
Vol 7 (7) ◽  
pp. 520
Author(s):  
Jianmin Fu ◽  
Nohelli E. Brockman ◽  
Brian L. Wickes
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Yeast Cell ◽  
Transformation Efficiency ◽  
Transformation Frequency ◽  
Yeast Cells ◽  
Agar Concentration ◽  
Genetic Tool ◽  
Number Of Factors ◽  
Cryptococcus Spp ◽  
Transformation Systems

The transformation of Cryptococcus spp. by Agrobacterium tumefaciens has proven to be a useful genetic tool. A number of factors affect transformation frequency. These factors include acetosyringone concentration, bacterial cell to yeast cell ratio, cell wall damage, and agar concentration. Agar concentration was found to have a significant effect on the transformant number as transformants increased with agar concentration across all four serotypes. When infection time points were tested, higher agar concentrations were found to result in an earlier transfer of the Ti-plasmid to the yeast cell, with the earliest transformant appearing two h after A. tumefaciens contact with yeast cells. These results demonstrate that A. tumefaciens transformation efficiency can be affected by a variety of factors and continued investigation of these factors can lead to improvements in specific A. tumefaciens/fungus transformation systems.

scholarly journals MicroRNA Zma-miR528 Versatile Regulation on Target mRNAs during Maize Somatic Embryogenesis

2021 ◽  
Vol 22 (10) ◽  
pp. 5310
Author(s):  
Eduardo Luján-Soto ◽  
Vasti T. Juárez-González ◽  
José L. Reyes ◽  
Tzvetanka D. Dinkova
Keyword(s):  
Somatic Embryogenesis ◽  
Immature Embryo ◽  
Inverse Correlation ◽  
Total Rna ◽  
Target Mrnas ◽  
Sequence Complementarity ◽  
Low Levels ◽  
Embryogenic Callus Induction ◽  
Regenerated Plantlets ◽  
Polysome Fraction

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the accumulation and translation of their target mRNAs through sequence complementarity. miRNAs have emerged as crucial regulators during maize somatic embryogenesis (SE) and plant regeneration. A monocot-specific miRNA, mainly accumulated during maize SE, is zma-miR528. While several targets have been described for this miRNA, the regulation has not been experimentally confirmed for the SE process. Here, we explored the accumulation of zma-miR528 and several predicted targets during embryogenic callus induction, proliferation, and plantlet regeneration using the maize cultivar VS-535. We confirmed the cleavage site for all tested zma-miR528 targets; however, PLC1 showed very low levels of processing. The abundance of zma-miR528 slightly decreased in one month-induced callus compared to the immature embryo (IE) explant tissue. However, it displayed a significant increase in four-month sub-cultured callus, coincident with proliferation establishment. In callus-regenerated plantlets, zma-miR528 greatly decreased to levels below those observed in the initial explant. Three of the target transcripts (MATE, bHLH, and SOD1a) showed an inverse correlation with the miRNA abundance in total RNA samples at all stages. Using polysome fractionation, zma-miR528 was detected in the polysome fraction and exhibited an inverse distribution with the PLC1 target, which was not observed at total RNA. Accordingly, we conclude that zma-miR528 regulates multiple target mRNAs during the SE process by promoting their degradation, translation inhibition or both.

scholarly journals Plant regeneration and transformation of Trachyspermum ammi using Agrobacterium tumefaciens and zygotic embryos

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Masoumeh Nomani ◽  
Masoud Tohidfar
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Plant Regeneration ◽  
Genetic Transformation ◽  
Zygotic Embryo ◽  
Plant Biotechnology ◽  
Hplc Method ◽  
Zygotic Embryos ◽  
Naphthaleneacetic Acid ◽  
Transformed Plants ◽  
Trachyspermum Ammi

Abstract Background Trachyspermum ammi is one of the key medicinal plant species with many beneficial properties. Thymol is the most important substance in the essential oil of this plant. Thymol is a natural monoterpene phenol with high anti-microbial, anti-bacterial, and anti-oxidant properties. Thymol in the latest research has a significant impact on slowing the progression of cancer cells in human. In this research, embryos were employed as convenient explants for the fast and effectual regeneration and transformation of T. ammi. To regenerate this plant, Murashige and Skoog (MS) and Gamborg's B5 (B5) media were supplemented with diverse concentrations of plant growth regulators, such as 6-benzyladenine (BA), 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and kinetin (kin). Transgenic Trachyspermum ammi plants were also obtained using Agrobacterium-mediated transformation and zygotic embryos explants. Moreover, two Agrobacterium tumefaciens strains (EHA101 and LBA4404) harboring pBI121-TPS2 were utilized for genetic transformation to Trachyspermum ammi. Results According to the obtained results, the highest plant-regeneration frequency was obtained with B5 medium supplemented with 0.5 mg/l BA and 1 mg/l NAA. The integrated gene was also approved using the PCR reaction and the Southern blot method. Results also showed that the EHA101 strain outperformed another strain in inoculation time (30 s) and co-cultivation period (1 day) (transformation efficiency 19.29%). Furthermore, HPLC method demonstrated that the transformed plants contained a higher thymol level than non-transformed plants. Conclusions In this research, a fast protocol was introduced for the regeneration and transformation of Trachyspermum ammi, using zygotic embryo explants in 25–35 days. Our findings confirmed the increase in the thymol in the aerial part of Trachyspermum ammi. We further presented an efficacious technique for enhancing thymol content in Trachyspermum ammi using Agrobacterium-mediated plant transformation system that can be beneficial in genetic transformation and other plant biotechnology techniques.

Agrobacterium tumefaciens-mediated Transformation of Double Haploid Canola (Brassica napus) Lines

1997 ◽  
Vol 24 (1) ◽  
pp. 97 ◽  
Author(s):  
K. Kazan ◽  
M. D. Curtis ◽  
K. C. Goulter ◽  
J. M. Manners
Keyword(s):  
Brassica Napus ◽  
Agrobacterium Tumefaciens ◽  
Transgenic Plants ◽  
Genetic Transformation ◽  
Double Haploid ◽  
Gene Promoter ◽  
Root Explants ◽  
Hypocotyl Explants ◽  
Peroxidase Gene ◽  
Hypocotyl Segments

Double haploid (DH) genotypes of canola (Brassica napus L.) have a high level of genetic uniformity but have not been previously tested for genetic transformation. Transgenic plants from three of four DH genotypes derived from cv. Westar were obtained by inoculation of either hypocotyl segments or root explants with Agrobacterium tumefaciens. For hypocotyl transformation, A. tumefaciens strain LBA4404 containing a binary plasmid with the neomycin phosphotransferase gene (nptII) and a CaMV 35S-peroxidase gene cassette was co-cultivated with hypocotyl segments taken from the 5–6-day-old seedlings. Transformation frequencies for hypocotyl explants of two DH genotypes were 0.3–3%. Direct evidence for genetic transformation of hypocotyl explants was obtained through molecular hybridisation analysis. Using this protocol, mature transformed plants were obtained within 4–6 months of co-cultivation. A method of root transformation was successfully modified for one DH genotype of canola and transgenic plants were obtained at a frequency of 2%. Using this protocol, a peroxidase gene promoter–GUS fusion construct was introduced into a DH genotype. Tissue specific GUS expression driven by the peroxidase gene promoter in transgenic plants was analysed by GUS staining. Transformation systems for double haploid canola lines will permit the assessment of introduced genes for their effect on agronomic and physiological traits.

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