scholarly journals 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)

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.

scholarly journals Successful genetic transformation in date palm (Phoenix dactylifera)

2014 ◽  
Vol 11 (2) ◽  
pp. 171-176 ◽  
Author(s):  
L Hassan
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Agrobacterium Rhizogenes ◽  
Reporter Gene ◽  
Hairy Roots ◽  
Date Palm ◽  
Binary Vector ◽  
Nptii Gene ◽  
Neomycin Phosphotransferase ◽  
Gus Gene ◽  
Nos Terminator

The introduction of foreign genes into most of the Phoenix spp using recombinant DNA technology is not a straight forward task. In Phoenix spp application of this technology towards successful transformation proved to be a more difficult one – so far no report on the successful regeneration of transgenic date palm plants has been published. We developed an efficient and reproducible variety-independent method for producing transgenic date palm (Phoenix spp) via Agrobacterium-mediated transformation. Agrobacterium rhizogenes strains LBA 9402 were used and for cotransformation experiments the strain LBA 9402 with the binary vector pBIN19 with the p35S GUS INT gene was used. Off-shoot segments from different Phoenix spp cultivars were infected with Agrobacterium rhizogenes. The development of ‘hairy roots’ at a high frequency only on infected tissue pieces showed that transformation is possible. Various parameters like, effect of different genotypes on root initiation, root number and root length have been studied. Regeneration of transformed root cultures to plantlets was also attempted. Histochemical GUS assay and polymerase chain reaction analysis of hairy roots confirmed the presence of GUS gene. Agrobacterium tumifaciensmediated transformation was also performed using the leaves of off-shoot explants. Agrobacterium tumefaciens strains: I) GV3101 with the vir plasmid pMP90 the strain C58C1 ATHV with the vir-plasmid pTiBo542 (=pEHA101; Hood et al. 1986) was used. The nptII gene (neomycin phosphotransferase) was used as a selectable marker gene. The ?-Glucuronidase-gene (GUS-Gene: Jefferson et al. 1987) under control of the Ubi- and 35S-Promotors, with an Intron (Vancanneyt et al. 1990), was used as the reporter gene. We also used the genetically engineered Agrobacterium tumefaciens strain LBA4404 as a vector for infection in the transformation experiment, which contains plasmid pBI121 of 14 KDa (binary vector). This binary vector contains following genes within the right border (RB) and left border (LB) region of the construct: The udiA gene (Jefferson, 1986) predetermining GUS (?-glucuronidase), driven by CaMV promoter and NOS terminator. This reporter gene can be used to assess the efficiency of transformation. The nptII gene (Herrera-Estrella et al., 1983) encoding neomycin phosphotransferase II (nptII) conferring kanamycin resistance, driven by NOS promoter and NOS terminator. The bacterium also contains plasmid pAL4404 which is a disarmed Ti plasmid (132 KDa) containing the virulence genes. For the confirmation of transgenes, calli were taken from the growing callus mass for DNA isolation. PCR- and Southern analysis was performed to determine the integration and the copy number of the transgene. The GUS-test was performed to demonstrate ß-glucuronidase expression. The transgenic plantlets were kept in a hardening room for four weeks and they will be transferred to a growth chamber with controlled environment for further establishment. DOI: http://dx.doi.org/10.3329/jbau.v11i2.19841 J. Bangladesh Agril. Univ. 11(2): 171-176, 2013

scholarly journals Regeneration and Transformation via Agrobacterium tumefaciens of Echinacea purpurea L.

1970 ◽  
Vol 20 (2) ◽  
pp. 101-111 ◽  
Author(s):  
Moemen Hanafy ◽  
Usama I. Aly ◽  
Mohamed A. Matter
Keyword(s):  
Selectable Marker ◽  
Binary Vector ◽  
Stem Elongation ◽  
Leaf Explants ◽  
Echinacea Purpurea ◽  
Plant Genome ◽  
Bar Gene ◽  
Herbicide Resistant ◽  
Glufosinate Ammonium ◽  
Chitinase Genes

Leaf explants of Echinacea purpurea L. taken from aseptically germinated seedlings were inoculated with A. tumefaciens strains EHA105, carrying a binary vector conferring herbicide resistant bar gene and fungal resistant chitinase gene. Glufosinate ammonium-resistant shoots were regenerated on a medium containing BAP and NAA at a concentration of 4.88 and 0.053 µM, respectively. A subsequent transfer of shoots to medium containing BAP was necessary for stem elongation and leaf development. Transgenic Echinacea plants carrying bar and chitinase genes were selected for their resistance to glufosinate ammonium herbicide. Molecular analysis using PCR confirmed the integration of the transgenes into plant genome. This is the first report on genetic transformation of Echinacea plant using bar gene as a selectable marker. Key words: Echinacea purpurea; Regeneration; A. tumefaciens; Transformation D.O.I. 10.3329/ptcb.v20i2.6888 Plant Tissue Cult. & Biotech. 20(2): 101-111, 2010 (December)

Generation of transgenic Eucalyptus globulus plantlets through Agrobacterium tumefaciens mediated transformation

1998 ◽  
Vol 25 (2) ◽  
pp. 207 ◽  
Author(s):  
M. Moralejo ◽  
F. Rochange ◽  
A.M. Boudet ◽  
C. Teuliéres
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Eucalyptus Globulus ◽  
Transformation Efficiency ◽  
Target Material ◽  
Selective Medium ◽  
Molecular Evidence ◽  
Factors Affecting ◽  
Gusa Gene ◽  
Mature Seeds ◽  
Transgenic Eucalyptus

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.

scholarly journals Agrobacterium-Mediated Stable Transformation of Medicinal Plant Andrographis paniculata Callus Expressing β-glucuronidase (GUS) Gene

2015 ◽  
Vol 18 (2) ◽  
pp. 92
Author(s):  
Erly Marwani ◽  
Agustina Tangapo ◽  
Fenny Martha Dwivany
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Gus Expression ◽  
Selective Medium ◽  
Stable Transformation ◽  
Andrographis Paniculata ◽  
Pcr Analysis ◽  
Induction Medium ◽  
Gus Gene ◽  
Glucuronidase Activity ◽  
Leaf Disks

This study was carried out to establish a stable genetic transformation in callus culture of Andrographispaniculata mediated by Agrobacterium tumefaciens. The leaf disks of A. paniculata were infected with A. tumefaciensLBA4404 carrying a binary vector pCAMBIA1304 that contain β-glucuronidase (GUS) and hygromycinphosphotransferase (hpt) genes. The infection was conducted by dipping method for one hour, followed byco-cultivation in the dark for three days. To examine transient GUS expression, the co-cultivated leaf disks wereassayed for β-glucuronidase activity and to obtain stable transformed callus, the co-cultivated leaf disks wereselected on the callus induction medium which contain 20 mg/l hygromycin for selection. The transformedcallus was periodically subcultured every three weeks into the fresh selection medium over the 15 weeksperiod. To test a stable transformation, the callus was subjected to PCR analysis for GUS gene detection. Theresults indicated that the co-cultivated leaf disks expressed GUS activity and proliferated to produce callus onthe selective medium. Analysis of PCR on the transformed callus indicated the presence 976 bp fragment thatconfi rmed the presence of β-glucuronidase gene. These fi ndings imply that the β-glucuronidase was stably integrated into A. paniculata callus culture.Keywords: Andrographis paniculata, Agrobacterium tumefaciens, andrographollide, transformed callus,β-glucuronidase gene.

scholarly journals Regeneration and transformation of Polish cultivars of potato

2014 ◽  
Vol 64 (4) ◽  
pp. 335-340
Author(s):  
Anna Nadolska-Orczyk ◽  
Lidia Miłkowska ◽  
Andrzej Pałucha ◽  
Paweł Czembor ◽  
Wacław Orczyk
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Transgenic Plants ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Transformation Efficiency ◽  
Binary Vector ◽  
Leaf Explants ◽  
Vector System ◽  
Regeneration Ability

The article presents the results of regeneration and transformation experiments of 12 Polish cultivars of potato. The cultivars Brda, Bzura, Elipsa and Irga regenerated the highest number of shoots from leaf explants (60% to 100% of explants regenerated 4.9 to 16.5 shoots per explant). The cultivars Brda, Bzura, Elipsa and Irys were the source of the best regenerating tuber explants (66% to 100% of explants regenerated 6.2 to 11.9 shoots from one explant). Both types of explants (from leaves and tubers) were used for transformation experiments. <i>Agrobacterium tumefaciens</i> strains used for transformation contained binary vector system: LBA 4404 (pAL 4404:pBI 121) and C58C1 (pGV2260:pVU104). There was a strong correlation between regeneration ability of tested cultivars and transformation efficiency. GUS-positive, kanamycin resistant and well rooted plants from leaf explants in cultivars Bzura, Brda and from tuber explants in Bzura and Elipsa were obtained. Northern blot analysis confirmed the presence of β-glucuronidase mRNA in transgenic plants.

scholarly journals 605 Agrobacterium-mediated Cranberry (Vaccinium macrocarpon) Transformation

HortScience ◽  
2000 ◽  
Vol 35 (3) ◽  
pp. 501C-501
Author(s):  
Luping Qu ◽  
James Polashock ◽  
Nicholi Vorsa
Keyword(s):  
Transformation Efficiency ◽  
Selectable Marker ◽  
Binary Vector ◽  
Adventitious Shoots ◽  
Leaf Explants ◽  
Vaccinium Macrocarpon ◽  
Marker Genes ◽  
Regeneration Medium ◽  
Selectable Marker Genes ◽  
Gus Gene Expression

Putative transgenic cranberry plants have been achieved via Agrobacterium-mediated transformation. Leaf explants were transformed with a supervirulent Agrobacterium tumefaciens strain EHA 105, harboring the binary vector P35SGUSint and nptII selectable marker genes. Inoculation of precultured explants (≈10 days on regeneration medium) coupled with sonicasion improved transformation efficiency significantly. Adventitious shoots were directly regenerated from explants. Putative transformed shoots were identified by being kanamycin-resistant and GUS-positive. Stable GUS gene expression (turning blue) could be detected within 1 h of incubation at 37 °C. Confirmation of transformation by molecular analysis is in progress. Eight putative transgenic cranberry plants were obtained. All appeared morphologically normal. This appears to be the first success in achieving cranberry transformed plants by Agrobacterium-mediated method. Optimizing the transformation system is ongoing.

scholarly journals Transformation of `Beurre Bosc' Pear with the rolC Gene

1999 ◽  
Vol 124 (6) ◽  
pp. 570-574 ◽  
Author(s):  
Richard L. Bell ◽  
Ralph Scorza ◽  
Chinnathambi Srinivasan ◽  
Kevin Webb
Keyword(s):  
Enzyme Activity ◽  
Agrobacterium Tumefaciens ◽  
Binary Vector ◽  
Leaf Explants ◽  
Rolc Gene ◽  
Proliferation Medium ◽  
Reduced Height ◽  
Kanamycin Selection ◽  
Selection Medium

`Beurre Bosc' pear (Pyrus communis L.) was transformed with Agrobacterium tumefaciens (E.F. Smith & Townsend) Conn strain EHA101 containing the binary vector pGA-GUSGF into which the rolC gene had been inserted. Leaf explants from in vitro shoot tip cultures were wounded, Agrobacterium-inoculated, and cultured on kanamycin selection medium. Regenerating shoots were transferred to proliferation medium without antibiotics. Three clones tested positive for GUS and nptII enzyme activity. Transformation with the rolC gene was confirmed by DNA, RNA, and protein blot analyses. The number of copies of the rolC transgene varied from one to three. Plantlets of the three transgenic clones were acclimated and transferred to the greenhouse. Preliminary observations of phenotype indicate that the rolC gene reduced height, number of nodes, and leaf area of transgenic `Beurre Bosc'.

scholarly journals Agrobacterium tumefaciens-transient genetic transformation of Habanero pepper (Capsicum chinense Jacq.) leaf explants

2010 ◽  
Vol 13 (4) ◽  
Author(s):  
Guadalupe Fabiola Arcos-Ortega ◽  
Rafael Antonio Chan-Kuuk ◽  
Wilma Aracely González-Kantún ◽  
Ramón Souza-Perera ◽  
Yumi Elena Nakazawa-Ueji ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Leaf Explants ◽  
Capsicum Chinense ◽  
Habanero Pepper
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