Generation of transgenic Eucalyptus globulus plantlets through Agrobacterium tumefaciens mediated transformation

A procedure for genetic transformation of Eucalyptus globulus Labill. using Agrobacterium tumefaciens is described. Young seedlings obtained from mature seeds were chosen as target material. The most important factors affecting transformation efficiency were the nature of the A. tumefaciens strain, precultivation of the seedlings and wounding of the tissues. The optimised procedure allowed us to obtain the first transgenic plants for E. globulus. On selective medium, 1.2% of the inoculated seedlings regenerated shoots resistant to kanamycin and expressing the gusA gene. Molecular evidence for the presence of stably integrated T-DNA is provided for two plants.

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Factors affecting transformation efficiency of embryogenic callus of Upland cotton (Gossypium hirsutum) with Agrobacterium tumefaciens

Plant Cell Tissue and Organ Culture (PCTOC) ◽

10.1007/s11240-004-5209-9 ◽

2005 ◽

Vol 81 (2) ◽

pp. 229-237 ◽

Cited By ~ 74

Author(s):

Shuangxia Jin ◽

Xianlong Zhang ◽

Shaoguang liang ◽

Yichun Nie ◽

Xiaoping Guo ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Gossypium Hirsutum ◽

Embryogenic Callus ◽

Upland Cotton ◽

Transformation Efficiency ◽

Factors Affecting

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An Improved Protocol for Agrobacterium-Mediated Transformation in Subterranean Clover (Trifolium subterraneum L.)

International Journal of Molecular Sciences ◽

10.3390/ijms22084181 ◽

2021 ◽

Vol 22 (8) ◽

pp. 4181

Author(s):

Fernando Perez Rojo ◽

Sumedha Seth ◽

William Erskine ◽

Parwinder Kaur

Keyword(s):

Agrobacterium Tumefaciens ◽

Nutritive Value ◽

Transformation Efficiency ◽

Incubation Temperature ◽

Agronomic Traits ◽

Genetic Manipulation ◽

Subterranean Clover ◽

Trifolium Subterraneum ◽

Factors Affecting

Subterranean clover (Trifolium subterraneum) is the most widely grown annual pasture legume in southern Australia. With the advent of advanced sequencing and genome editing technologies, a simple and efficient gene transfer protocol mediated by Agrobacterium tumefaciens was developed to overcome the hurdle of genetic manipulation in subterranean clover. In vitro tissue culture and Agrobacterium transformation play a central role in testing the link between specific genes and agronomic traits. In this paper, we investigate a variety of factors affecting the transformation in subterranean clover to increase the transformation efficiency. In vitro culture was optimised by including cefotaxime during seed sterilisation and testing the best antibiotic concentration to select recombinant explants. The concentrations for the combination of antibiotics obtained were as follows: 40 mg L−1 hygromycin, 100 mg L−1 kanamycin and 200 mg L−1 cefotaxime. Additionally, 200 mg L−1 cefotaxime increased shoot regeneration by two-fold. Different plant hormone combinations were tested to analyse the best rooting media. Roots were obtained in a medium supplemented with 1.2 µM IAA. Plasmid pH35 containing a hygromycin-resistant gene and GUS gene was inoculated into the explants with Agrobacterium tumefaciens strain AGL0 for transformation. Overall, the transformation efficiency was improved from the 1% previously reported to 5.2%, tested at explant level with Cefotaxime showing a positive effect on shooting regeneration. Other variables in addition to antibiotic and hormone combinations such as bacterial OD, time of infection and incubation temperature may be further tested to enhance the transformation even more. This improved transformation study presents an opportunity to increase the feeding value, persistence, and nutritive value of the key Australian pasture.

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Development of Efficient, Reproducible and Stable Agrobacterium-Mediated Genetic Transformation of Five Potato Cultivars

Food Technology and Biotechnology ◽

10.17113/ftb.58.01.20.6187 ◽

2020 ◽

Vol 58 (1) ◽

pp. 57-63

Author(s):

Allah Bakhsh

Keyword(s):

Transformation Efficiency ◽

Transgenic Potato ◽

Factors Affecting ◽

Leaf Discs ◽

Potato Plants ◽

Gus Histochemical Assay ◽

Regenerated Plants ◽

Transgenic Potato Plants ◽

Gusa Gene ◽

Primary Transformants

The developments in transformation technology have enabled the scientists to incorporate, mutate or substitute gene(s) leading to a particular trait; advancing it to a point where only few technical limitations remain. Genotype dependency and explant types are important factors affecting transformation efficiency in potato. In the present study, a rapid, reproducible and stable Agrobacterium-mediated transformation procedure in potato was developed by a combination of different plant growth regulators. Leaf discs and internodal explants of five cultivars of potato, i.e. Lady Olympia, Granola, Agria, Désirée and Innovator were infected with Agrobacterium tumefaciens strain LBA4404 containing pBIN19 expression vector with β-glucuronidase gusA gene under the control of 35S CaMV promoter. Kanamycin was used as plant selectable marker for screening of primary transformants at concentration of 100 mg/L. Both explants responded positively; internode being more suitable explant for better transformation efficiency. Based on GUS histochemical assay, the transformation efficiency was 22, 20, 18.6, 15 and 10 % using the internodal explant, and 15, 12, 17, 8 and 6 % using leaf discs as explant in Lady Olympia, Granola, Agria, Désirée and Innovator respectively. Furthermore, PCR assays confirmed the presence of gusA and nptII genes in regenerated plants. The molecular analysis in succeeding progeny showed proper integration and expression of both genes. The results suggest Lady Olympia as the best cultivar for future transformation procedures. Overall, the short duration, rapidity and reproducibility make this protocol suitable for wider application of transgenic potato plants.

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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)

JURNAL BIOS LOGOS ◽

10.35799/jbl.2.1.2012.375 ◽

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.

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An improved Agrobacterium tumefaciens mediated transformation of Artemisia annua L. by using stem internodes as explants

Czech Journal of Genetics and Plant Breeding ◽

10.17221/105/2012-cjgpb ◽

2013 ◽

Vol 49 (No. 3) ◽

pp. 123-129 ◽

Cited By ~ 3

Author(s):

N. Tian ◽

S. Liu ◽

H. Ting ◽

J. Huang ◽

S. van der Krol ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Optical Density ◽

Culture Medium ◽

Root Formation ◽

Transformation Efficiency ◽

Artemisia Annua ◽

Adventitious Shoot ◽

Selective Medium ◽

Artemisia Annua L ◽

Low Efficiency

Transformation of Artemisia annua, which produces the sesquiterpenoid endoperoxide artemisinin widely used for the treatment of malaria, has been hampered by the low efficiency of adventitious shoot and root formation on a selective medium containing additional compounds for Agrobacterium decontamination. Here we identified several factors which were all shown to be of importance for optimization of Artemisia annua transformation. Results indicated that stem internodes showed better resistance capacity to Agrobacterium decontaminator than leaves did. Agrobacterium tumefaciens with an optical density (OD) value of 0.2–0.5 plus 100 μmol of acetosyringone per litre of solution gave the best transformation efficiency. Moreover, kanamycin at 30 mg/l in the culture medium was effective in suppressing the growth of non-transformed tissue. Furthermore, transgenic shoots required an early induction of rooting. In addition, dimethyl sulphoxide considerably improved the rooting of shoots. The present work provides rapid and reproducible transformation and regeneration of A. annua.

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

Journal of Fungi ◽

10.3390/jof7070520 ◽

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.

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Multi-Drug Resistant Organisms Infection Impact on Patients Length of Stay in Respiratory Care Ward

Antibiotics ◽

10.3390/antibiotics10050608 ◽

2021 ◽

Vol 10 (5) ◽

pp. 608

Author(s):

Yi-Ping Chen ◽

Xian-Wen Tasi ◽

Ko Chang ◽

Xuan-Di Cao ◽

Jung-Ren Chen ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Length Of Hospital Stay ◽

Selective Medium ◽

Respiratory Care ◽

Control Group ◽

Resistant Organism ◽

Case Group ◽

Drug Resistant ◽

Factors Affecting ◽

Care Ward

This study aimed to investigate the effects of multi-drug-resistant organism (MDRO) infection and other factors on the length of hospital stay (LOS) of patients in the respiratory care ward (RCW) of a regional hospital in Taiwan. In this retrospective study, we collected cases from MDRO-infected patients in the RCW from January 2016 to March 2020. The RCW comprises 13 beds in total. There were 106 infected patients, of which 42 were in the case group (infected with MDROs) and 64 were in the control group (not infected with MDROs). Clinical specimens were inoculated in a selective medium to isolate the pathogenic bacteria by standard procedures. The results showed the main factors affecting the LOS were: patients with MDRO infection, patients discharged from the RCW, and patients who underwent catheterization. The LOS of patients infected with MDROs was significantly longer than that of patients without MDRO infection (β = 0.55, 95% CI = 0.02–1.09), with the case group and the control group being 479.8 ± 546.5 and 307.3 ± 436.2 days, respectively. Infection with carbapenem-resistant Pseudomonas aeruginosa (CRPA) was associated with a longer LOS than other MDRO strains. These findings have important implications for infection control in RCW and in better tracking the health of patients.

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