Production of Transgenic Plants via Agrobacterium tumefaciens- Mediated Genetic Transformation in Pinus wallichiana (Himalayan Blue Pine (original) (raw)
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Agrobacterium tumefaciens-Mediated Genetic Transformation of Pinus kesiya Royle ex Gord (Khasi Pine
This study highlights, for first time, an Agrobacterium-mediated gene transfer method for the genetic improvement of Pinus kesiya. The genetic transformation of P. kesiya has been limited by difficulty in selection efficiencies and a low transformation frequency. Embryo-genic cultures were established from zygotic embryos according to our previous protocol (Malabadi et al. 2005). During transformation events, rapidly growing embryogenic tissue of three genotypes were co-cultivated with disarmed A. tumefaciens strain EHA105 fused with a binary vector pBI121, which contains the neomycin phosphotransferase II (nptII) gene providing kanamycin resistance as a selectable marker and the-glucuronidase (uidA) reporter gene, was used in the transformation studies. All the transgenic lines exhibited very low maturation potential compared to the control. GUS activity was used to monitor transient expression of the uidA gene and to further test lines selected on kanamycin-containing medium. The integration of one of the transgenes, nptII, was confirmed by PCR followed by Southern and Northern blot analyses. Agrobacterium-mediated gene transfer was found to be a very useful technique for large-scale generation of transgenic P. kesiya, and may prove useful for other recalcitrant conifer species.
Stable and Consistent Agrobacterium-Mediated Genetic Transformation in Pinus roxburghi (Chir Pine
This paper highlights an Agrobacterium tumefaciens-mediated transformation protocol, developed for embryogenic cell cultures derived from vegetative shoot apices of mature, 14 years-old trees of Pinus roxburghii. The plasmid pBI121, containing the neomycin phosphotransferase II (nptII) gene providing kanamycin resistance as a selectable marker and the-glucuronidase (uidA/GUS) reporter gene, was used as a binary vector. Transformation frequencies were dependent on the species, genotype and post-cocultivation procedure. The highest transformation efficiency was obtained in the embryogenic line PR105 (37 transformed lines/g fresh wt) than with the embryogenic lines PR11 and PR521. The transgenic state of the embryogenic tissue was initially confirmed by histochemical GUS assay. Stable integration of the nptII gene in the plant genome of P. roxburghii was confirmed by polymerase chain reaction (PCR), Southern and Northern blot analyses. These results demonstrated that a stable and enhanced transformation system has been established in chir pine, and that this system would provide an opportunity to transfer economically important genes into other genotypes of P. roxburghii.
An Agrobacterium-mediated transformation procedure was developed to transform the mature embryo from Pinus monticola (Dougl. ex D. Don) seeds with two binary vectors containing the reporter gene encoding the green fluorescent protein (GFP) or the-glucuronidase protein (GUS), respectively. More than 1000 embryos from independent transformation events were tested for different western white pine seed families. Selection of kanamycin-resistant callus tissues showed that survival rates varied from 33 to 48% in different independent experiments. Transgenic callus tissues survived and continued to grow on the medium with kanamycin (25 g/mL), whereas non-transgenic callus, regenerated from the embryos of the same seed family, died within 12 weeks. Integration and expression of the introduced reporter gene was confirmed in transgenic western white pine calli by GUS-staining analysis or microscopic observation of GFP fluorescence. Rates for stable reporter gene expression ranged from 2.9 to 6.5% for all embryos co-cultured with Agrobacterium. Our protocol has enabled the routine transformation of western white pine, a species that was previously difficult for gene manipulation. To our knowledge, this is the first report on genetic engineering of this conifer. Our results demonstrate that transgenic gene expression in western white pine is a feasible option for genetic improvement of this valuable conifer as well as for investigating its molecular interactions with the fungal pathogen Cronartium ribicola (J.C. Fisch.).
1999
A genetic transformation procedure for white pine has been developed after cocultivation of embryogenic tissues with Agrobacterium tumefaciens. This efficient transformation procedure led to an average of four independent transformed lines per gram of cocultivated embryogenic tissue and up to 50 transformed lines can be obtained in a routine experiment. Constructs bearing the uidA gene or the green fluorescent protein (GFP) gene were introduced and β-glucuronidase (GUS) activity was followed over time. The expression of the uidA gene was lowest with a 35S-gus-intron construct and was 20-fold higher with a 35S-35S-AMVgus::nptII construct. The addition of scaffold attachment region (SAR) sequences surrounding the gus::nptII fusion did not significantly enhance the GUS activity. Transformed mature somatic embryos have been germinated and plantlets are presently being acclimatized.
Planta, 2001
Embryos of 24 open-pollinated families of loblolly pine (Pinus teade L.) were used as explants to conduct in vitro regeneration. Then, Agrobacterium tumefaciens strain GV3101 harboring the plasmid pPCV6NFHygGUSINT was used to transform mature zygotic embryos of seven families of loblolly pine. The frequency of transformation varied among families infected with A. tumefaciens. The highest frequency (100%) of transient beta-glucuronidase (GUS)-expressing embryos was obtained from family 11-1029 with over 300 blue spots per embryo. Expression of the GUS reporter gene was observed in cotyledons, hypocotyls, and radicles of co-cultivated mature zygotic embryos, as well as in callus and shoots derived from co-cultivated mature zygotic embryos. Ninety transgenic plants were regenerated from hygromycin-resistant callus derived from families W03. 8-1082 and 11-1029. and 19 transgenic plantlets were established in soil. The presence of the GUS gene in the plant genome was confirmed by polymer...
Production of transgenic Phalaenopsis plants using Agrobacterium tumefaciens
1999
Transgenic Phalaenopsis plants over-expressing glutathione S-transferase (GST) gene were produced by Agrobacterium-mediated transformation method. Protocorms at an early stage after germination were inoculated with A. tumefaciens strain EHA101 (pEKH35S163P) that harbors GST, hygromycin phosphotransferase and neomycin phosphotransferase II genes. A total of 68 independent transgenic plants derived from 6325 mature seeds were obtained 6 months after infection with Agrobacterium. Regenerated plants were grown in greenhouse and all plants bloomed within 2 years. Five months after pollination, 13 seed pods were obtained from 6 plants showing suitable commercial characters. Germinated Phalaenopsis T1 seedlings with GST gene were confirmed by PCR analysis. Northern blot analysis confirmed the expression of GST in hygromycin-resistant plantlets.
Towards genetic engineering of maritime pine (Pinus pinaster Ait.)
Annals of forest science, 2002
Using our improved protocols for somatic embryogenesis in Pinus pinaster, transgenic tissues and plantlets were recovered after microprojectile bombardment (biolistic) or cocultivation of embryonal-suspensor masses (ESM) with Agrobacterium tumefaciens. Transformation experiments were carried out with selectable hpt gene (hygromycin B resistance) and reporter gus gene (β-glucuronidase activity). With both methods, hygromycin was shown to be an effective selective agent of transformed cells within 4-19 weeks. The mean number of hygromycin-resistant lines expressing gus per gram ESM subjected to DNA transfer, ranged from 7.0 to 8.5 using biolistic and 0 to 67.3 during Agrobacterium experiments. Mature somatic embryos obtained from some transformed lines were converted into plantlets and grown in the greenhouse. The whole process (from transformation to plant acclimatisation) could be completed within only 12 months. The transgenic state of ESM, somatic embryos and plants was confirmed by histochemical GUS assays and molecular methods. Pinus pinaster / somatic embryogenesis / biolistic / Agrobacterium tumefaciens / transgenic plant Résumé-Transformation génétique du pin maritime (Pinus pinaster Ait.). En appliquant nos protocoles d'embryogenèse somatique développés pour Pinus pinaster, des tissus et plantes transgéniques ont été obtenus après bombardement avec des microparticules (biolistique) ou coculture de masses embryonnaires (ESM) avec Agrobacterium tumefaciens. Les expériences de transformation ont été conduites à l'aide du gène de sélection hpt (résistance à l'hygromycine B) et du gène rapporteur gus (activité β-glucuronidase). L'hygromycine a permis de sélectionner efficacement les cellules transformées par ces deux méthodes en 4 à 19 semaines. Le nombre moyen de lignées résistantes à l'hygromycine exprimant le gène gus obtenu par gramme d'ESM varie de 7,0 à 8,5 (biolistique) ou de 0 à 67,3 (Agrobacterium). Les embryons matures obtenus à partir de certaines de ces lignées ont pu être convertis en plantules élevées en serre. Seulement 12 mois sont nécessaires de la transformation des ESM jusqu'à l'acclimatation des plantes. La nature transgénique des ESM, embryons somatiques et plantes, a été confirmée à l'aide de tests histochimiques « GUS » et de méthodes moléculaires.
Plant Cell Reports, 2010
A protocol was developed for genetic transformation of somatic embryos derived from juvenile and mature Quercus robur trees. Optimal transformation conditions were evaluated on the basis of the results of transient GUS expression assays with five oak embryogenic lines and a strain of Agrobacterium tumefaciens (EHA105) harbouring a p35SGUSINT plasmid containing a nptII and a uidA (GUS) genes. For stable transformation, embryo clumps at globular/torpedo stages (4–10 mg) were inoculated with EHA105:p35SGUSINT bacterial cultures, cocultivated for 4 days and selected in proliferation medium with 75 mg/l of kanamycin. Putatively transformed masses appeared after 20–30 weeks of serial transfers to selective medium. Histochemical and molecular analysis (PCR and Southern blot) confirmed the presence of nptII and uidA genes in the plant genomes. Transformation efficiencies ranged from up to 2% in an embryogenic line derived from a 300-year-old tree, to 6% in a juvenile genotype. Twelve independent transgenic lines were obtained from these oak genotypes, and transgenic plantlets were recovered and acclimatized into the soil. This is the first demonstration of the production of transformed somatic embryos and regenerated plants from juvenile and mature trees of Q. robur and suggests the possibility of introducing other genetic constructions to develop trees that are tolerant/resistant to pathogens and/or biotic stresses.
Agrobacterium-mediated DNA transfer in sugar pine
Plant Molecular Biology, 1990
DNA transfer using Agrobacterium tumefaciens has been demonstrated in sugar pine, Pinus lambertiana Dougl. Shoots derived from cytokinin-treated cotyledons formed galls after inoculation with A. tumefaciens strains containing the plasmid pTiBo542. A selectable marker, neomycin phosphotransferase II, conferring resistance to kanamycin, was transferred into sugar pine using a binary armed vector system. Callus proliferated from the galls grew without hormones and in some cases, kanamycin-resistant callus could be cultured. Southern blots provided evidence of physical transfer of T-DNA and the nptlI gene. Expression of the nptlI gene under control of the nos promoter was demonstrated by neomycin phosphotransferase assays. Several aspects of DNA transfer were similar to those previously observed in angiosperms transformed by A. tumefaciens. This is the first evidence for DNA transfer by Agrobacterium in this species and the first physical evidence for transfer in any pine. These results bring us closer to genetic engineering in this commercially important genus of forest trees.
Optimization of Agrobacterium-mediated genetic transformation in gherkin (Cucumis anguria L.)
Plant Omics, 2013
The present work was aimed to study various factors influencing Agrobacterium tumefaciens mediated genetic transformation of gherkin (Cucumis anguria L). Agrobacterium strain LBA4404 harboring binary vector pBAL2 carrying the reporter gene βglucuronidase intron (gus) and the marker gene neomycin phosphotransferase (nptII) was used for transformation. Factors affecting transformation efficiency, such as Agrobacterium concentration, effect of acetosyringone, pre-cultivation, infection and cocultivation time of Agrobacterium were studied. After co-cultivation, explants were transferred into MS medium plus B5 vitamins (MSB 5 ) containing 1.5 μM benzylaminopurine (BAP) with 0.5 μM naphthalene acetic acid (NAA), 100 mg L -1 kanamycin and 300 mg L -1 carbenicillin for callus induction. Regeneration of adventitious shoots from callus was achieved on MSB 5 medium containing 3.0 μM BAP, 100 mg L -1 kanamycin and 300 mg L -1 carbenicillin. Transgenic shoots were elongated in MSB 5 medium fortified with 2.0 μM gibberellic acid (GA 3 ), 100 mg L -1 kanamycin and 300 mg L -1 carbenicillin. The transgenic elongated shoots were rooted in MSB 5 medium supplemented with 3.0 μM indole 3-butyric acid (IBA) and 100 mg L -1 kanamycin. The putative transgenic plants were acclimatized in the greenhouse. A strong β-glucuronidase activity was detected in the transformed plants by histochemical assay. Integration of T-DNA into the nuclear genome of transgenic plants was confirmed by polymerase chain reaction and southern hybridization. The nptII gene expression in transgenic plants was confirmed by RT-PCR. A transformation efficiency of 15% was obtained. This protocol allows effective transformation and direct regeneration of C. anguria.