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.

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 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

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 In vitro Regeneration of Dalbergia sissoo Roxb. and the Potential for Genetic Transformation

2012 ◽  
Vol 40 (2) ◽  
pp. 140 ◽  
Author(s):  
Hafiz Mamoon REHMAN ◽  
Iqrar Ahmad RANA ◽  
Siddra IJAZ ◽  
Ghulam MUSTAFA ◽  
Faiz Ahmad JOYIA ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Genetic Transformation ◽  
Callus Induction ◽  
In Vitro Regeneration ◽  
Induction Medium ◽  
Native Forest ◽  
Ms Medium ◽  
Dalbergia Sissoo ◽  
Callus Induction Medium

Dalbergia sissoo Roxb. ex DC. (Sissoo) is a native forest tree species in Pakistan. Many ecological and economical uses are associated with this premier timber species, but dieback disease is of major concern. The objective of this study was to develop a protocol for in vitro regeneration of Sissoo that could serve as target material for genetic transformation, in order to improve this species. Callus formation and plantlet regeneration was achieved by culturing cotyledons, immature seeds, and mature embryos on a modified Murashige and Skoog (1962) (MS) medium supplemented with plant growth regulators. Callus induction medium containing 2.71 ?M 2, 4-dichlorophenoxyacetic acid (2,4-D) and 0.93 ?M kinetin produced better callus on all explants tested compared to other treatments, such as 8.88 ?M 6-benzylaminopurine (BA) and 2.69 ?M ?-naphthalene acetic acid (NAA), or 2.71 ?M 2, 4-D and 2.69 ?M NAA. Shoot regeneration was best on MS medium containing 1.4 ?M NAA and 8.88 ?M BA compared to other treatments, such as 1.4 ?M NAA and 9.9 ?M kinetin, or 2.86 ?M indole-3-acetic acid and 8.88 ?M BA. Murashige and Skoog medium containing 1.4 NAA ?M and 8.88 ?M BA was better in general for regeneration regardless of callus induction medium and the type of explant used. Rooting was best on half-strength MS medium with 7.35 ?M indole-3-butyric acid. Regenerated plantlets were acclimatized for plantation in the field. Preliminary genetic transformation potential of D. sissoo was evaluated by particle bombardment of callus explants with a pUbiGus vector. The bombarded tissue showed transient Gus activity 1week after bombardment. Transformation of this woody tree is possible provided excellent regeneration protocols. The best combination for regeneration explained in this study is one of such protocols.

scholarly journals Kanamycin in Cereal Biotechnology: A Screening or a Selectable Marker?

2021 ◽  
Vol 3 (1) ◽  
pp. 01-05
Author(s):  
Malik Shuja
Keyword(s):  
Transgenic Plants ◽  
Genetic Transformation ◽  
Selectable Marker ◽  
Normal Growth ◽  
Minimal Effect ◽  
Effective Screening ◽  
Transformed Plants ◽  
Transformation Systems ◽  
Selection Agent ◽  
Screening Marker

Kanamycin is a widely used selection agent in dicot-plant genetic transformation systems. In monocots, however, it does not seem to be effective as it has no or minimal effect on the normal growth of non-transformed plants. Kanamycin was previously demonstrated to bleach the pigments of the non-transgenic plants. This may yield the idea that kanamycin can be used as an effective screening marker rather than a selectable marker in monocots.

scholarly journals Improvement of plant regeneration and Agrobacterium-mediated genetic transformation of Stylosanthes guianensis

2019 ◽  
Vol 7 (5) ◽  
pp. 480-492 ◽  
Author(s):  
Pengfei Guo ◽  
Pandao Liu ◽  
Jian Lei ◽  
Caihong Chen ◽  
Hong Qiu ◽  
...  
Keyword(s):  
Plant Regeneration ◽  
Genetic Transformation ◽  
Function Analysis ◽  
Acid Soils ◽  
Limiting Factors ◽  
Pcr Analysis ◽  
Induction Medium ◽  
Ms Medium ◽  
Stylosanthes Guianensis ◽  
2,4 Dichlorophenoxyacetic Acid

As a pioneer tropical pasture legume, stylo (Stylosanthes guianensis) is well adapted to growth-limiting factors in acid soils. Considering the importance of stylo, there is a need to improve Agrobacterium-mediated genetic transformation to enable development of elite cultivars. In this study, S. guianensis cv. RY5 was used to systematically optimize Agrobacterium-mediated transformation based on its plant regeneration. Results showed that Murashige and Skoog (MS) medium containing 0.2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 2 mg/L 6-benzylaminopurine (6-BA) was the optimal callus induction medium. MS medium supplemented with 2 mg/L 6-BA was suitable for shoot regeneration from cotyledon-derived calluses, and 0.5 mg/L indole-3-acetic acid (IAA) and 0.5 mg/L indole-3-butyric acid (IBA) applications were beneficial for rooting. The highest transformation efficiency (67%) was obtained at an Agrobacterium concentration of optical density = 0.6 combined with an infection time of 15 min and 3 days of co-cultivation. Furthermore, 200 mg/mL carbenicillin (Carb) and 0.6 mg/L Basta® supplements were effective in eliminating excess bacterial growth and selecting transgenic plants, respectively. Subsequent polymerase chain reaction (PCR) analysis confirmed that the β-glucuronidase (GUS) and BAR genes were successfully integrated into the stylo genome. Wider testing of this improved protocol as a means of enhancing genetic improvement and gene function analysis of stylo seems warranted.

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 Kanamycin in Cereal Biotechnology: A Screening or a Selectable Marker?

2021 ◽  
Vol 3 (1) ◽  
pp. 01-05
Author(s):  
Malik Shuja ◽  
Hassan Riaz ◽  
Muhsin Jamal ◽  
Muhammad Imran
Keyword(s):  
Transgenic Plants ◽  
Genetic Transformation ◽  
Selectable Marker ◽  
Normal Growth ◽  
Minimal Effect ◽  
Effective Screening ◽  
Transformed Plants ◽  
Transformation Systems ◽  
Selection Agent ◽  
Screening Marker

Kanamycin is a widely used selection agent in dicot-plant genetic transformation systems. In monocots, however, it does not seem to be effective as it has no or minimal effect on the normal growth of non-transformed plants. Kanamycin was previously demonstrated to bleach the pigments of the non-transgenic plants. This may yield the idea that kanamycin can be used as an effective screening marker rather than a selectable marker in monocots.   Copyright (c) 2021 Malik Nawaz Shuja, Hasan Riaz, Muhsin Jamal, Muhammad Imran

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.

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