Agrobacterium tumefaciens-Mediated Genetic Transformation of Green Gram [Vigna radiata (L.) Wilczek] - A Recalcitrant Grain Legume

2021 ◽  
Author(s):  
Aparna Priyadarshini Patra ◽  
Kailash Chandra Samal ◽  
Gyana Ranjan Rout ◽  
Simachal Sahu ◽  
Prem Narayan Jagadev
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Vigna Radiata ◽  
In Vitro Regeneration ◽  
Cotyledonary Node ◽  
Green Gram ◽  
Grain Legume ◽  
Marker Genes ◽  
Soil Productivity ◽  
Ms Medium

Background: Green gram is grown in many parts of India as a source of dietary protein (21-25%). It is an important nitrogen fixing crop which fixes atmospheric nitrogen (119-140 kg/ha) to soil and enhance the soil productivity. In the present investigation, efficient Agrobacterium-mediated genetic transformation of Vigna radiata L. (Wilczek) has been achieved with VrTIP1 gene for abiotic stress resistance i.e. moisture and salinity stress. Methods: Four days old shoot tip and cotyledonary node were used for in vitro regeneration with MS medium supplemented with BAP 2.0 mg/l, kinetin 0.5 mg/l and 50 mg/l kanamycin for co-cultivation with Agrobacterium tumefaciens strains, LBA 4404. The modified binary vector pCXSN, EHA105 containing hygromycin phosphotransferase II (hpt II) marker genes and a synthetic TIP1 gene under a constitutive CaMV35S promoter were used for transformation of Vigna radiata L. cotyledonary node explants. Putative transformants selected from hygromycin resistant shoots were subsequently rooted on MS medium supplemented with 1.0 mg/l NAA and later transferred to sterile vermiculite followed by transfer to the transgenic green house. Result: The T1 plants were produced from PCR positive T0 plants and analysed for presence and integration of transgenes in putative T1 plants were confirmed by polymerase chain reaction (PCR) amplification of 752 bp of hpt II fragment. This protocol can be effectively used for transferring new traits in greengram and other legumes for their quantitative and qualitative improvements.

Agrobacterium tumefaciens-mediated genetic transformation of mungbean (Vigna radiata L. Wilczek) — a recalcitrant grain legume

Plant Science ◽  
2001 ◽  
Vol 161 (2) ◽  
pp. 239-247 ◽  
Author(s):  
Pawan K. Jaiwal ◽  
Ragini Kumari ◽  
S. Ignacimuthu ◽  
Ingo Potrykus ◽  
Christof Sautter
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Vigna Radiata ◽  
Grain Legume

Somatic embryogenesis and plant regeneration from cotyledonary explants of green gram [Vigna radiata (L.) Wilezek.]—A recalcitrant grain legume

2006 ◽  
Vol 42 (2) ◽  
pp. 134-138 ◽  
Author(s):  
C. P. Kaviraj ◽  
G. Kiran ◽  
R. B. Venugopal ◽  
P. B. Kavi Kishor ◽  
Srinath Rao
Keyword(s):  
Somatic Embryogenesis ◽  
Plant Regeneration ◽  
Vigna Radiata ◽  
Green Gram ◽  
Grain Legume ◽  
Cotyledonary Explants

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 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 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 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.

In vitro regeneration and Agrobacterium tumefaciens-mediated genetic transformation of D. lotus ( Diospyros lotus L.)

2018 ◽  
Vol 236 ◽  
pp. 229-237 ◽  
Author(s):  
Xuehan Li ◽  
Zhenying Jiang ◽  
Yanying Shen ◽  
Feihong Li ◽  
Xinyi Yu ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
In Vitro Regeneration ◽  
Diospyros Lotus

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 genetic transformation of a recalcitrant grain legume, lentil (Lens culinaris Medik)

2008 ◽  
Vol 28 (3) ◽  
pp. 407-417 ◽  
Author(s):  
Ufuk Celikkol Akcay ◽  
M. Mahmoudian ◽  
H. Kamci ◽  
M. Yucel ◽  
H. A. Oktem
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Lens Culinaris ◽  
Grain Legume
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