scholarly journals GENETIC TRANSFORMATION AND REGENERATION OF TRANSGENIC SWEETPOTATO PLANTS.

HortScience ◽  
1995 ◽  
Vol 30 (3) ◽  
pp. 435f-435 ◽  
Author(s):  
Marceline Egnin ◽  
C.S. Prakash
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Transgenic Plants ◽  
Genetic Transformation ◽  
Binary Vector ◽  
Blot Hybridization ◽  
Pcr Amplification ◽  
Ms Medium ◽  
Petiole Explants ◽  
Efficient Delivery ◽  
Foreign Genes

This study aimed to optimize factors for the efficient delivery of foreign genes into sweetpotato using Agrobacterium tumefaciens and develop transgenic plants. Disarmed Agrobacterium C58 carrying a binary vector pBI 121C2H with gusA, nptll, and the nutritional protein asp-l genes was used to cocultivate (4 days) petiole explants of the sweetpotato genotype P1318846-3. Pre-incubation of petioles for 3 days on MS medium with 2,4-D (0.2 mg·liter–1) before infection resulted in higher transformation. Putative transgenic shoots were obtained by transfer of petioles to MS medium with TDZ (0.2 mg·liter–1) and kanamycin (80 to 140 mg·liter–1). The PCR amplification of gusA, nptll, and asp-1 genes in the 37 putative transgenic shoots showed that six plants contained the three genes. However, none of these plants showed histochemical expression of the gusA gene. The introduced gene may have been methylated resulting in the lack of its expression. DNA blot hybridization studies are underway to verify the presence and integration of the transgenes.

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.

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.

scholarly journals Agrobacterium-mediated genetic transformation of two varieties of Brassica: optimization of protocol

1970 ◽  
Vol 34 (2) ◽  
pp. 287-301 ◽  
Author(s):  
MMA Khan ◽  
ABMAHK Robin ◽  
MAN Nazim-Ud-Dowla ◽  
SK Talukder ◽  
L Hassan
Keyword(s):  
Brassica Napus ◽  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Brassica Rapa ◽  
Binary Vector ◽  
Gus Assay ◽  
Key Words Transformation ◽  
Gus Reporter ◽  
Best Response ◽  
Transformation Experiment

 Two rapeseed varieties, namely Tori-7 and BARI Sarisha-8, respectively, from Brassica rapa and Brassica napus were selected to observe the transformation ability. Petioles were inoculated in Agrobacterium tumefaciens strain LBA 4404 carrying a binary vector pBl2l with GUS (reporter) and nptII (kanamycin resistant) gene. The transformation experiment was performed by optimizing two important factors: preculture time and co-cultivation time and also selected out the best variety. Infection was most effective when explants were pre-cultured for 72 hours (80% GUS positive). and co-cultivated for 72 hours (72% GUS positive). The variety Tori-7 showed the best response to GUS assay (65% GUS positive). Callus induction was the highest in Tori-7, which were 6% with 72 hours of preculture period and 9% in 48 hours of co-cultivation. Number of putative transformed plantlets were highest in Tori-7 (7 plants) followed by BARI Sarisha-8 (3 plants). Key words: Transformation; Brassica; GUS; Agrobacterium. DOI: 10.3329/bjar.v34i2.5802Bangladesh J. Agril. Res. 34(2): 287-301, June 2009

scholarly journals Agrobacterium-mediated Transformation of Cotyledon Explants of Bottle Gourd (Lagenaria siceraria S.)

HortScience ◽  
2004 ◽  
Vol 39 (4) ◽  
pp. 821E-822
Author(s):  
Jeung-Sul Han* ◽  
Chang Kil Kim
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Binary Vector ◽  
Bottle Gourd ◽  
Infection Frequency ◽  
Ms Medium ◽  
Lagenaria Siceraria ◽  
Cultivation Medium ◽  
Glufosinate Ammonium ◽  
Cotyledon Explants ◽  
Selection Medium

A procedure for producing transgenic bottle gourd plants by inoculating cotyledon explants with Agrobacterium tumefaciens strain AGL1 carrying a binary vector pCAMBIA3301, which contains glufosinate ammonium-resistant (bar) and the reporter (gus) genes, is describe. Infection was the most effective (highest infection frequency and index) when explants were co-cultivated with Agrobacterium for 6-8 days on co-cultivation medium supplemented with 0.001-0.1 mg/L L-a-(2-aminoethoxyvinyl) glycine (AVG). Transgenic plants were obtained with frequencies of about 0.2% when the explants were cultured on selection medium (MS medium supplemented with 3.0 mg/L BAP, 0.5 mg/L AgNO3, 500 mg/L cefotaxime, 2.0 mg/L DL-phosphinothricin, 0.3% sucrose and 0.8% Plant Agar. A histochemical gus assay, PCR and Southern blot analyses confirmed that transformation had occurred. Genetic analysis of T1 progenies showed that the transgenes were inherited in a Mendelian fashion. To our knowlege, this study represents the first report for Agrobacterium-mediated transformation in bottle gourd, rootstock for watermelon and other cucurbit crops in many countries.

scholarly journals Improvement of adventitious organogenesis for regeneration of transgenic plants in blackberry

Genetika ◽  
2015 ◽  
Vol 47 (2) ◽  
pp. 599-608 ◽  
Author(s):  
Alena Gajdosová ◽  
Tatjana Vujovic ◽  
Miroslava Súkeníková ◽  
Gabriela Libiaková
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Plant Regeneration ◽  
Gene Transfer ◽  
Transgenic Plants ◽  
Plant Genome ◽  
Ms Medium ◽  
Regeneration System ◽  
Promising Technique ◽  
Rubus Fruticosus ◽  
Foreign Dna

The introduction of foreign DNA into the plant genome by Agrobacterium tumefaciens is a promising technique of targeted gene transfer which depends on good working regeneration system. The aim of the work was to elaborate the system for efficient adventitious organogenesis and transgenic plant regeneration in Rubus fruticosus L. using explants from mature plants. Regeneration of putative transgenic shoots took place from flag explants cultivated vertically on MS medium with 1 mg l-1 TDZ and 0.02 mg l-1 IBA followed by transfer on MS medium with 1 mg l-1 BAP, 0.02 mg l-1 IBA and 0.1 mg l-1 GA3 supplemented with 10-15 mg l-1 hygromycin after transformation by A. tumefaciens strain LBA 4404 carrying plasmid pCambia 1304. Four putative transgenic plants of cv. 'Cacanska Bestrna' were rooted and acclimatized.

scholarly journals Agrobacterium tumefaciens-mediated transformation of Jatropha curcas L. with a polyhydroxyalkanoate gene (phaC)

2018 ◽  
Vol 22 (2) ◽  
pp. 61 ◽  
Author(s):  
Chesara Novatiano ◽  
Adi Pancoro ◽  
Erly Marwani
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Jatropha Curcas ◽  
Ralstonia Eutropha ◽  
Plant Biomass ◽  
Bacterial Biomass ◽  
Ms Medium ◽  
Phac Gene ◽  
Selection Medium ◽  
Pha Genes

Polyhydroxybutyrate is a component of bioplastics that is synthesized under the control of enzymes encoded by pha multigenes. The genes are naturally present in Ralstonia eutropha. However, the production of bioplastics in bacteria is inefficient because the bacterial biomass is relatively small compared with plants or fungi. As such, engineering techniques have been developed that enable pha genes to be inserted into plant biomass, and then be expressed in the biomass of the plant to produce polyhydroxybutyrate. The objectives of this study were to transform the tissue of Jatropha curcas using the phaC gene (a pha gene), to regenerate the transformed plant, and to confirm the presence of the inserted genes with PCR. The genetic transformation of J. curcas was mediated by Agrobacterium tumefaciens strain GV3101 containing pARTC by dipping the cotyledon tissue of J. curcas in a suspension of the bacterium for 30 min, followed by cocultivation for 3 d on Murashige and Skoog (MS) medium. The tissue was then placed on a selection medium, i.e. MS medium containing 13.3 µM BAP and 0.05 µM IBA with the addition of 20 mg/L kanamycin. The results showed that 12.35% of the tissue survived and regenerated into a shoot after 1–2 months. Molecular analysis of the transformed tissue was performed using phaC and nptII primers, in order to detect the presence of the phaC and nptII genes. Specific bands were detected at 659 bp and 700 bp, corresponding to the nptII primer and phaC primer, respectively.

Regeneration of vgb-transgenic poplar (Populus alba×P. glandulosa) and the primary observation of growth

2006 ◽  
Vol 3 (1) ◽  
pp. 59-64 ◽  
Author(s):  
Zhang Bing-Yu ◽  
Su Xiao-Hua ◽  
Li Yi-Liang ◽  
Huang Qin-Jun ◽  
Zhang Xiang-Hua ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Blot Hybridization ◽  
Pcr Amplification ◽  
First Year ◽  
Populus Alba ◽  
Rt Pcr ◽  
Dot Blot ◽  
Transgenic Lines ◽  
Polymerase Chain ◽  
Morphological Differences

AbstractIncreasing the growth rate is especially important for low-quality wood applications, so this has become an important goal in poplar breeding. The present study describes the transfer of Vitreoscilla haemoglobin (VHb) gene (vgb) driven by constitutive promoters, by Agrobacterium tumefaciens into poplar (Populus alba×P. glandulosa). From about 450 leaf discs used for transformation, 60 Kan-resistant plants were obtained, and 52 proved to be true transgenic plants. The transgenic nature of these plants was confirmed by polymerase chain reaction (PCR) amplification and Southern dot blot hybridization. The expression of vgb gene in transgenic plants was confirmed by reverse transcriptase-PCR (RT-PCR). The performance of the transgenic lines was evaluated during the first year of growth in a greenhouse. These plants showed no significant stable morphological differences from the untransformed plants. Among them, three vgb-transgenic lines exhibited noticeably higher growth rates in terms of height and diameter.

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