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 Transformation of Nicotiana alata Link and Otto. with an Autoregulatory Senescence-inhibitor Gene Construct

HortScience ◽  
1996 ◽  
Vol 31 (4) ◽  
pp. 617c-617
Author(s):  
Kenneth R. Schroeder ◽  
Dennis P. Stimart
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Leaf Explants ◽  
Shoot Proliferation ◽  
Nicotiana Alata ◽  
Ms Medium ◽  
Gene Encoding ◽  
Half Strength ◽  
Cytokinin Synthesis ◽  
Senesced Leaves

Leaf explants of Nicotiana alata Link and Otto. were surface disinfested and cultured on Murashige and Skoog (MS) medium containing 2.66 μm N6-benzyladenine (BA) to promote shoot proliferation. After 5 weeks, proliferated shoots were removed and remaining callus saved. Callus was inoculated with Agrobacterium tumefaciens encoding a senescence-specific promoter SAG12 cloned from Arabidopsis thaliana fused to a Agrobacterium tumefaciens gene encoding isopentenyl transferase which catalyzes cytokinin synthesis. Following inoculation, the callus was cocultivated for 6 days on BA medium. Selection for transgenics was done on BA medium plus 100 mg Kanamycin and 400 mg Ticarcillin (antibiotics) per liter. Proliferating shoots were rooted on MS medium containing antibiotics. Rooted cuttings were transplanted to soil, acclimated and flowered in the greenhouse. Transgenics were outcrossed to a commercial N. alata hybrid. Seed was germinated in vitro on half-strength MS medium plus antibiotics. Segregation of transgenics to nontransgenics was 1:1. Evaluation of leaf senescence on 5-month-old plants showed 2 to 14 times fewer senesced leaves on the transgenic than the nontransgenic plants.

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 Ekspresi fenotipe gen APETALA1 kakao (TcAP1) pada eksplan tembakau Phenotypic expression of cacao APETALA1 (TcAP1) in tobacco explant

2016 ◽  
Vol 74 (1) ◽  
Author(s):  
Tetty CHAIDAMSARI ◽  
. SAMANHUDI ◽  
Asmini BUDIANI ◽  
Roedhy POERWANTO ◽  
Djoko SANTOSO
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Tobacco Plant ◽  
Phenotypic Expression ◽  
Plant Cells ◽  
Leaf Explants ◽  
Leaf Disk ◽  
Rt Pcr ◽  
Set Up ◽  
Morphological Phenotype

Summary APETALA1 (AP1) is one of flowering identity genes that determines the formation of sepal and petal tissues. An AP1 homologue was cloned from cacao flowers by bio-techniques coupled with bio-informatics. Examination of phenotypic expression was conducted with transgenesis of the 35S-TcAP1 construct using leaf disk technique of tobacco leaf explants mediated by Agrobacterium tumefaciens. PCR specific to TcAP1 demonstrated that the technique is effective in introducing the 35S-TcAP1 construct into tobacco plant cells. RT-PCR with total RNA from the leaves of transgenic tobacco plantlets showed that expression levels of the TcAP1 events varied. The variation of the transcript levels was comparable to the morphological phenotype of the tobacco plantlets grown in vitro. The cultures expressing TcAP1 at moderate levels, have developed into intact plantlets and set up flowers in vitro.Ringkasan APETALA1 (AP1) diketahui merupakan salah satu gen identitas pembungaan yang mengendalikan terbentuknya jaringan sepal dan petal. Homolog AP1 telah diklon dari organ bunga kakao (TcAP1) dengan kombinasi bio-techniques dan bio-informatics. Pengujian ekspresi fenotipe TcAP1 dilakukan dengan transgenesis konstruk konstitutif 35S-TcAP1 menggunakan teknik leaf disk eksplan daun tembakau dan mediasi Agrobacterium tumefaciens. Pengujian PCR spesifik TcAP1 menunjukkan bahwa teknik tersebut cukup efektif dalam mengintroduksikan konstruk 35S-TcAP1 ke dalam sel tanaman tembakau. RT-PCR dari daun planlet tembakau trangenik membuktikan bahwa tingkat ekspresi TcAP1 tersebut bervariasi. Perbedaan level ekspresi TcAP1 ini memberikan pengaruh yang nampak sebanding terhadap perkembangan morfologis planlet tembakau in vitro.  Kultur yang mengekspresikan TcAP1 pada level sedang mampu beregenerasi menjadi planlet sempurna dan membentuk bunga in vitro.

scholarly journals An Evaluation of Antibiotics for the Elimination of Agrobacterium tumefaciens from Apple Leaf Explants in Vitro and for the Effect on Regeneration

HortScience ◽  
1995 ◽  
Vol 30 (4) ◽  
pp. 876C-876 ◽  
Author(s):  
F.A. Hammerschlag ◽  
R.H. Zimmerman ◽  
U.L. Yadava ◽  
S. Hunsucker ◽  
P. Gercheva
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Malus Domestica ◽  
Leaf Explants ◽  
Vacuum Infiltration ◽  
Short Term

A range of antibiotics was evaluated for their effect on eliminating Agrobacterium tumefaciens supervirulent strain EHA101(pEHA101) from leaf explants of `Royal Gala' apple (Malus domestica Borkh) and on regeneration. After long-term (38 days) exposure to 100-μg–ml–1 concentrations of either cefotaxime (cef), carbenicillin (carb), mefoxin (mef), or combinations of these antibiotics, only on carb or carb with mef was regeneration not inhibited. None of the above antibiotics or antibiotic combinations eliminated A. tumefaciens from leaf explants. Short-term (1-18 hours), vacuum infiltration with 500- to 1000-μg–ml–1 concentrations of either of the above antibiotics did not inhibit regeneration, but did not eliminate A. tumefaciens from leaf explants. After a 30-min vacuum infiltration with a 2000-μg–ml–1 concentration of either cef, carb, or mef, only cef reduced the number of leaf explants with A. tumefaciens.

Interactions of Agrobacterium tumefaciens with soybean (Glycine max (L.) Merr.) leaf explants in tissue culture

1986 ◽  
Vol 28 (5) ◽  
pp. 808-817 ◽  
Author(s):  
D. T. Kudirka ◽  
S. M. Colburn ◽  
M. A. Hinchee ◽  
M. S. Wright
Keyword(s):  
Tissue Culture ◽  
Glycine Max ◽  
Agrobacterium Tumefaciens ◽  
Culture Medium ◽  
Leaf Explants ◽  
Wound Response ◽  
Ti Plasmid ◽  
Tissue Culture Medium ◽  
Glycine Max L

Wounded petiole surfaces of excised soybean (Glycine max (L.) Merr.) leaves of 'Peking' formed neoplastic growths testing positive for opines after inoculation with a tumorigenic strain of Agrobacterium tumefaciens. During the 48-h inoculation period, uninoculated explants and explants inoculated with A136 (avirulent), A208 (tumorigenic), and ASE1-200 (nontumorigenic) strains of A. tumefaciens showed initiation of mitotic activity at the wound surface conforming with classical descriptions of the wound response; however, shortly after explant transfer to tissue culture medium, morphogenetic patterns in explants inoculated with tumorigenic A. tumefaciens deviated from the uninoculated and A136 inoculated controls. This developmental deviation was correlated to the presence of the Ti plasmid in the bacterium. Explants inoculated with nontumorigenic A. tumefaciens showed a morphogenetic pattern intermediate between the controls and explants inoculated with tumorigenic bacteria. This suggests that the latter plant response might be due to a plant regulator gene found in the vir region of the Ti plasmid. Explants inoculated with tumorigenic bacteria showed decreasing efficiency of transformation if inoculations were delayed 4 h after excision and explants showed greatest thermosensitivity to transformation during the 48-h inoculation period. Reduced thermosensitivity was evident after transfer to tissue culture medium supplemented with carbenicillin. In this in vitro inoculation procedure, 'Peking' was shown to be twice as transformable as 'Corsoy 79'. This offers an opportunity to determine if there is a heritable component associated with transformation in this soybean explant system.Key words: Agrobacterium tumefaciens, Glycine, transformation, wound response, tissue culture.

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 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 Ekspresi fenotipe gen APETALA1 kakao (TcAP1) pada eksplan tembakau Phenotypic expression of cacao APETALA1 (TcAP1) in tobacco explant

2016 ◽  
Vol 74 (1) ◽  
Author(s):  
Tetty CHAIDAMSARI ◽  
. SAMANHUDI ◽  
Asmini BUDIANI ◽  
Roedhy POERWANTO ◽  
Djoko SANTOSO
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Tobacco Plant ◽  
Phenotypic Expression ◽  
Plant Cells ◽  
Leaf Explants ◽  
Leaf Disk ◽  
Rt Pcr ◽  
Set Up ◽  
Morphological Phenotype

Summary APETALA1 (AP1) is one of flowering identity genes that determines the formation of sepal and petal tissues. An AP1 homologue was cloned from cacao flowers by bio-techniques coupled with bio-informatics. Examination of phenotypic expression was conducted with transgenesis of the 35S-TcAP1 construct using leaf disk technique of tobacco leaf explants mediated by Agrobacterium tumefaciens. PCR specific to TcAP1 demonstrated that the technique is effective in introducing the 35S-TcAP1 construct into tobacco plant cells. RT-PCR with total RNA from the leaves of transgenic tobacco plantlets showed that expression levels of the TcAP1 events varied. The variation of the transcript levels was comparable to the morphological phenotype of the tobacco plantlets grown in vitro. The cultures expressing TcAP1 at moderate levels, have developed into intact plantlets and set up flowers in vitro.Ringkasan APETALA1 (AP1) diketahui merupakan salah satu gen identitas pembungaan yang mengendalikan terbentuknya jaringan sepal dan petal. Homolog AP1 telah diklon dari organ bunga kakao (TcAP1) dengan kombinasi bio-techniques dan bio-informatics. Pengujian ekspresi fenotipe TcAP1 dilakukan dengan transgenesis konstruk konstitutif 35S-TcAP1 menggunakan teknik leaf disk eksplan daun tembakau dan mediasi Agrobacterium tumefaciens. Pengujian PCR spesifik TcAP1 menunjukkan bahwa teknik tersebut cukup efektif dalam mengintroduksikan konstruk 35S-TcAP1 ke dalam sel tanaman tembakau. RT-PCR dari daun planlet tembakau trangenik membuktikan bahwa tingkat ekspresi TcAP1 tersebut bervariasi. Perbedaan level ekspresi TcAP1 ini memberikan pengaruh yang nampak sebanding terhadap perkembangan morfologis planlet tembakau in vitro.  Kultur yang mengekspresikan TcAP1 pada level sedang mampu beregenerasi menjadi planlet sempurna dan membentuk bunga in vitro.

scholarly journals An Agrobacterium mediated transformation system of guava (Psidium guajava L.) with endochitinase gene

2014 ◽  
Vol 14 (4) ◽  
pp. 232-237 ◽  
Author(s):  
Maneesh Mishra ◽  
Syed Uzma Jalil ◽  
Nimisha Sharma ◽  
Umesh Hudedamani
Keyword(s):  
Transformation Efficiency ◽  
Binary Vector ◽  
Nuclear Genome ◽  
Psidium Guajava ◽  
Nptii Gene ◽  
Cultivation Medium ◽  
Putative Transformants ◽  
Selection Medium ◽  
First Time

Genetic transformation of guava (Psidium guajava L.) was developed for the first time using in vitro grown shoot tip explant co-cultivated with Agrobacterium tumefaciens strain LBA4404 harbouring binary vector pIIHR-JBMch with endochitinase and nptII genes. The highest transformation efficiency was achieved by wounding explants with tungsten particles (0.5 µm) through particle acceleration system, followed by infection for 45 minutes with A. tumefaciens, grown overnight with 100 µM acetosyringone, corresponding to OD600=0.5 followed by co-cultivation for 72 hours under dark condition on co-cultivation medium (MS+100 µM acetosyringone+100 mg L-1 L-Cystein). Putative transformed explants regenerated shoots on selection medium stressed with 200 mg L-1 kanamycin for 12 weeks. Molecular analysis of putative transformants by PCR confirmed the integration of endochitinase and nptII gene in the plant nuclear genome.

Sign in / Sign up

Export Citation Format

Share Document