Agrobacterium-mediated in planta transformation of maize via pistil filaments

Integration of T-DNA into the maize genome as a result of treatment of silks with Agrobacterium cells, containing activated vir genes, was demonstrated. In planta treatment of maize (Zea mays L.) was performed during flowering in field. Cell suspension of Agrobacterium tumefaciens line GV3101(pTd33), carrying activated vir genes, was applied onto the previously isolated silks, which were afterwards pollinated with the pollen of the same cultivar. Integration of T-DNA into maize genome was confirmed by PCR (the nptII and gus reporter genes) and hystochemical staining of the seedling tissues, obtained from the transformed seeds. Amplification of the nptII gene showed the presence of about 60.3% of PCR-positive plants out of the total number of kanamycin-resistant seedlings examined, or 6.8% of the total of number of seedlings.     

Agrobacterium tumefaciens-mediated transformation of bush monkey-flower (Mimulus aurantiacus Curtis) with a new reporter gene ZsGreen

A successful in vitro Agrobacterium-mediated transformation protocol was developed for Mimulus aurantiacus, a model species for ecological and evolutionary genetics and a promising ornamental plant. Three binary vectors were tested, each containing the hptII selectable marker gene and one of the reporter genes: gusA, EGFP or ZsGreen, all of them under CaMV 35S promoter. Genetic transformation was achieved through 4 days of co-cultivation of leaf, petiole and hypocotyl explants with Agrobacterium tumefaciens strain LBA 4404. Explants produced transformed callus tissue on solid modified Murashige and Skoog medium supplemented with 1 mg L^?1 6-benzylaminopurine, 0.5 mg L^?1 1-naphthaleneacetic acid, 30 g L^?1 sucrose and 20 or 50 mg L^?1 hygromycin B. All three reporter genes were expressed in callus tissue but the intensity of expression gradually decreased during further plant development. The new reporter gene ZsGreen proved suitable for plant transformation experiments since very intense and bright fluorescence was detected. Out of 1,760 co-cultured explants, 110 plants were regenerated and all of them were found to be PCR positive for the selection and/or reporter genes. Chemiluminescent Southern blot analysis revealed that 91 % of the regenerated plants (100 T₀ plants) contained T-DNA integrated in their genome. Transformation efficiency varied from 1.4 to 23.3 % for hypocotyl and petiole explants, respectively. Integration of some backbone sequences in plant genomes was confirmed in 75.3 % of T₀ plants. Using this protocol, stable transformants expressing selectable marker gene hptII and one of the reporter genes (gusA, ZsGreen or EGFP) were obtained in 4–5 months.     

Cinnamic acid, coumarin and vanillin: Alternative phenolic compounds for efficient Agrobacterium-mediated transformation of the unicellular green alga, Nannochloropsis sp

The use of acetosyringone in Agrobacterium-mediated gene transfer into plant hosts has been favored for the past few decades. The influence of other phenolic compounds and their effectiveness in Agrobacterium-mediated plant transformation systems has been neglected. In this study, the efficacy of four phenolic compounds on Agrobacterium-mediated transformation of the unicellular green alga Nannochloropsis sp. (Strain UMT-M3) was assessed by using β-glucuronidase (GUS) assay. We found that cinnamic acid, vanillin and coumarin produced higher percentages of GUS positive cells as compared to acetosyringone. These results also show that the presence of methoxy group in the phenolic compounds may not be necessary for Agrobacterium vir gene induction and receptor binding as suggested by previous studies. These findings provide possible alternative Agrobacterium vir gene inducers that are more potent as compared to the commonly used acetosyringone in achieving high efficiency of Agrobacterium-mediated transformation in microalgae and possibly for other plants.     

Gene transfer into peanut (Arachis hypogaea L.) by Agrobacterium tumefaciens

Introduction of foreign genes into plant tissues via Agrobacterium tumefaciens based vectors requires specific knowledge of Agrobacterium-host compatibility. Therefore, to develop a transformation protocol for peanut (Arachis hypogaea L.), five Brazilian cultivars were screened with four wild-type A.tumefaciens strains. Successful transformation was dependent on specific bacterial strain-plant cultivar interactions and strain A281 was the most effective for tumor induction. Tumors displayed hormone autonomous growth, were opine positive and contained DNA that was homologous to the T-DNA of the inciting strain. Tumors induced on seed and seedling explants by A281 (pTD02) also expressed the reporter genes gus and npt-II contained in the binary vector. These results show that peanut is a permissive host for the acceptance of genes from specific A.tumefaciens gene vectors.Abbreviations GUS ß-glucuronidase (EC 3.2.1.31) - NPT-II neomycin phosphotransferase II (EC 2.7.1.95) - EDTA ethylene-diamine-tetracetic acid     

Absence in monocotyledonous plants of the diffusible plant factors inducing T-DNA circularization and vir gene expression in Agrobacterium

Summary T-DNA circularization is one of the molecular events specifically induced in agrobacterial cells upon their infection of dicotyledonous plant cells. We developed a seedling co-cultivation procedure to determine whether or not monocotyledonous plants have the ability to induce T-DNA circularization and vir gene expression. Co-cultivation of Agrobacterium tumefaciens with seedlings of dicotyledonous plants showed that the circularization event takes place efficiently. The exudates and extracts of the seedlings also effectively induced T-DNA circularization and vir gene expression, indicating that dicotyledonous seedlings contain diffusible factors capable of inducing these molecular events. In contrast, neither T-DNA circularization nor vir gene expression was detectable when Agrobacterium was incubated with seedlings of monocotyledonous plants. Supplementing with acetosyringone, a known inducer of vir gene expression and T-DNA circularization, resulted in the induction of circularization during co-cultivation with monocotyledonous seedlings. These results indicate that the seedlings of monocotyledonous plants have no detectable amounts of diffusible inducers, unlike dicotyledonous seedlings. Therefore, it is unlikely that the vir genes are expressed in Agrobacterium inoculated in monocotyledonous plants. This may be one of the blocks in tumorigenesis of monocotyledonous plants by Agrobacterium.     

Agrobacterium tumefaciens-mediated transformation of rutabaga (Brassica napus var. napobrassica) cultivar “American Purple Top Yellow”

An efficient protocol for genetic transformation of rutabaga (Brassica napus var. napobrassica) cultivar ??American Purple Top Yellow?? was developed by optimizing several factors influencing gene delivery and plant regeneration. A two-step regeneration protocol, adapted from canola, was optimal for rutabaga regeneration using hypocotyl explants. Transient expression studies monitored by histochemical ??-glucuronidase (GUS) assays indicated that several factors, including Agrobacterium tumefaciens strain, cocultivation time, and cocultivation medium, affected gene delivery. For stable transformation, precultured hypocotyl explants were cocultivated with Agrobacterium cells on sterilized filter paper overlaid on callus induction medium containing 100???M acetosyringone for 6?d under a 16-h photoperiod. Selection and regeneration of transformed cells were conducted on media containing 50?mg?l^?1 kanamycin and 250?mg?l^?1 Timentin. Using this protocol, GUS- and PCR-positive transformants were obtained from 3.2 to 4.2?% of hypocotyl explants inoculated with each of the three Agrobacterium strains after 3?C5?mo. Most transformants exhibited a normal phenotype. Southern blot analysis confirmed stable integration of the gusA transgene in T₀ plants.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated genetic transformation of a recalcitrant grain legume,lentil (<Emphasis Type="Italic">Lens culinaris</Emphasis> Medik)

A simple and reproducible Agrobacterium-mediated transformation protocol for a recalcitrant legume plant, lentil (Lens culinaris M.) is reported. Application of wounding treatments and efficiencies of three Agrobacterium tumefaciens strains, EHA105, C58C1, and KYRT1 were compared for T-DNA delivery into lentil cotyledonary node tissues. KYRT1 was found to be on average 2.8-fold more efficient than both EHA105 and C58C1 for producing transient β-glucuronidase (GUS) gene (gus) expression on cotyledonary petioles. Wounding of the explants, use of an optimized transformation protocol with the application of acetosyringone and vacuum infiltration treatments in addition to the application of a gradually intensifying selection regime played significant roles in enhancing transformation frequency. Lentil explants were transformed by inoculation with Agrobacterium tumefaciens strain, KYRT1 harboring a binary vector pTJK136 that carried neomycin phosphotransferase gene (npt-II) and an intron containing gusA gene on its T-DNA region. GUS-positive shoots were micrografted on lentil rootstocks. Transgenic lentil plants were produced with an overall transformation frequency of 2.3%. The presence of the transgene in the lentil genome was confirmed by GUS assay, PCR, RT-PCR and Southern hybridization. The transgenic shoots grafted on rootstocks were successfully transferred to soil and grown to maturity in the greenhouse. GUS activity was detected in vegetative and reproductive organs of T₀, T₁, T₂ and T₃ plants. PCR assays of T₁, T₂ and T₃ progenies confirmed the stable transmission of the transgene to the next generations.     

An efficient <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated transformation protocol of <Emphasis Type="Italic">Withania somnifera</Emphasis>

This is the first report on Agrobacterium rhizogenes-mediated transformation of Withania somnifera for expression of a foreign gene in hairy roots. We transformed leaf and shoot tip explants using binary vector having gusA as a reporter gene and nptII as a selectable marker gene. To improve the transformation efficiency, acetosyringone (AS) was added in three stages, Agrobacterium liquid culture, Agrobacterium infection and co-culture of explants with Agrobacterium. The addition of 75 μM AS to Agrobacterium liquid culture was found to be optimum for induction of vir genes. Moreover, the gusA gene expression in hairy roots was found to be best when the leaves and shoot tips were sonicated for 10 and 20s, respectively. Based on transformation efficiency, the Agrobacterium infection for 60 and 120 min was found to be suitable for leaves and shoot tips, respectively. Amongst the various culture media tested, MS basal medium was found to be best in hairy roots. The transformation efficiency of the improved protocol was recorded 66.5 and 59.5?% in the case of leaf and shoot tip explants, respectively. When compared with other protocols the transformation efficiency of this improved protocol was found to be 2.5 fold higher for leaves and 3.7 fold more for shoot tips. Southern blot analyses confirmed 1–2 copies of the gusA transgene in the lines W1-W4, while 1–4 transgene copies were detected in the line W5 generated by the improved protocol. Thus, we have established a robust and efficient A. rhizogenes mediated expression of transgene (s) in hairy roots of W. somnifera.     

Sonication-assisted efficient Agrobacterium-mediated genetic transformation of the multipurpose woody desert shrub Leptadenia pyrotechnica

The desert shrub Leptadenia pyrotechnica (Forssk.) Decne (Asclepiadaceae) is an important multipurpose woody species of tropical and sub-tropical arid regions. The shrub’s excellent pharmacological properties, importance in desert afforestation and role in sand dune fixation has been elaborately studied in recent years which make it a potential candidate for genetic manipulation in the global warming scenario. We have developed an Agrobacterium-mediated transformation protocol for L. pyrotechnica using hypocotyl explants from 5 days old seedlings. The reliability of the protocol has been tested by transforming the species with gus and gfp reporter genes separately. Hypocotyl explants were sonicated for 40 s, infected with Agrobacterium suspension of OD₆₀₀ 0.5, co-cultivated in the dark for 2 days at 26 °C in presence of 200 μM acetosyringone. Transgenic plants were obtained after 20–22 weeks at a frequency of 14 and 11 % for gus and gfp reporter genes respectively. Transgenic plants were confirmed by PCR and Southern blots. Expression of the two reporter genes has been tested in different stages of transgenic plant development. No phenotypic differences between the wild-type and transgenic plants were noted. This method will be very helpful to introduce alien genes-of-interest for various biotechnological applications.     

Agrobacterium tumefaciens-mediated transformation of Dendrobium lasianthera J.J.Sm: An important medicinal orchid

A protocol for genetic transformation mediated by Agrobacterium tumefaciens and production of transgenic Dendrobium lasianthera has been developed for the first time. The 8-week-old protocorm explants were used as target of transformation with Agrobacterium tumefaciens strain LBA4404 carrying plasmid pG35SKNAT1. Several parameters such as infection period, Agrobacterium density, concentration of acetosyringone, and co-cultivation period were evaluated for the transformation efficiency. The data were analyzed using one-way analysis of variance (ANOVA) and Duncan's Multiple Range Test (DMRT) with p?<?0.05. Subsequently, KNAT1 gene expression was confirmed by polymerase chain reaction (PCR) analysis. The highest efficiency of transformation (70%) obtained from protocorm explants infected with Agrobacterium culture was at the OD₆₀₀ concentration of 0.6 for 30?min, and co-cultivated with acetosyringone 100?µM for 5?days. The results of confirmation by PCR analysis show that the KNAT1 gene has been integrated and expressed in the genome of Dendrobium lasianthera transgenic.     

Transformation and Characterization of Transgenic Plants of Solanum dulcamara L.--Incidence of Transgene Silencing

A transformation system is described for Solanum dulcamara usingthe supervirulentAgrobacterium tumefaciens strain 1065, carryingboth the ß-glucuronidase (gus) and neomycin phosphotransferaseII (npt II) genes adjacent to the right and left T-DNA borders,respectively. Leaf explants were more efficient for the productionof transformed plants compared to stem explants on medium containing50 mg l^-1of kanamycin sulphate. A 1:10 (v:v) dilution of anovernight culture ofAgrobacterium gave optimal transformationin terms of transgenic plant regeneration. From a total of 174kanamycin-resistant plants selected by their antibiotic resistance,16 failed to exhibit GUS activity. Southern analysis revealedthat these GUS-negative transformants originated from threeindependently transformed cell lines. Restriction enzyme analysesshowed that the GUS-negative plants had both the gus and nptII genes integrated into their genome (one plant had a singlecopy of each gene; the other two plants had multiple copies),with major rearrangement of the gus gene occurring in plantswith several copies of the transgene. GUS-negative plants showedleaf malformations, delayed flowering and a reduction in flower,fruit and seed production compared to GUS-positive and non-transformed(control) plants. Although gene silencing of the gus gene occurred,albeit at a low frequency (9.2%), the transformation systemdescribed generates large numbers of phenotypically normal,stably transformed plants. Copyright 2000 Annals of Botany Company Agrobacterium -mediated transformation, gene silencing, Solanum dulcamara L. (Bittersweet, Woody Nightshade), T-DNA truncation, transgene expression     

Agrobacterium tumefaciens-mediated transformation of japonica and indica rice varieties

Genetic transformation of rice (Oryza sativa L.) mediated by Agrobacterium ttumefaciens has been confirmed for japonica varieties and extended to include the more recalcitrant indica varieties. Immature embryos were inoculated with either A. tumefaciens At656 (pCNL56) or LBA4404 (pTOK233). Experimental conditions were developed initially for immature embryos treated with strain At656, based upon both transient and stable -glucuromdase (GUS) activities. However, plant regeneration following selection on G418 (pCNL56 contained the nptII gene) did not occur. Using the same basic protocol, but inoculating immature embryos of rice with LBA4404 (pTOK233), resulted in efficient (about 27%) production of transgenic plants of the japonica variety, Radon, and an acceptable efficiency (from 1–5%) for the indica varieties IR72 and TCS10. Transformation was based upon resistance to hygromycin (pTOK233 contains the hpt gene), the presence of GUS activity (from the gusA gene), Southern blots for detection of the integrated gusA gene, and transmission of GUS activity to progeny in a Mendelian 3:1 segregation ratio. Southern blots indicated two to three copies of the gene integrated in most transformants. Transgenic plants of both the japonica and indica varieties were self-fertile and comparable in this respect to seed-grown plants. Key factors facilitating the transformation of rice by Agrobacterium tumefaciens appeared to be the use of embryos as the expiant, the use of hygromycin as the selection agent (which does not interfere with rice regeneration), the presence of extra copies of certain vir genes on the binary vector of pTOK233, and maintaining high concentrations of acetosyringone for inducing the vir genes during co-cultivation of embryos with Agrobacterium.Abbreviations AS acetosyringone - DMRT Duncan's Multiple Range Test - GUS -glucuronidase - T-DNA transferred DNA We wish to thank Dr. Toshihiko Komari, Japan Tobacco Inc. for providing Ayrobacterium tumefaciens strain LBA4404 (pTOK322). Support by the Rockefeller Foundation in the form of a fellowship to R.R.A. and a grant to T.K.H. is acknowledged. This is journal paper number 14,914 from the Purdue University Agricultural Experiment Station.     

A reliable and efficient protocol for inducing genetically transformed roots in medicinal plant Nepeta pogonosperma

Nepeta pogonosperma is an important medicinal plant with anti-inflammatory effects. An efficient and reliable transformation system for this plant was developed through optimization of several factors which affected the rate of Agrobacterium rhizogenes mediated transformation. Five bacterial strains, A4, ATCC15834, LBA9402, MSU440 and A13, two explant types, leaves and stems, and several co-cultivation media were examined. The maximum rate of hairy root induction was obtained from stem explants using MSU440 and ATCC15834 bacterial strains. A drastic increase in the frequency of transformation (91 %) was observed when MS medium lacking NH₄NO₃, KH₂PO₄, KNO₃ and CaCl₂. Hairy root lines were confirmed by polymerase chain reaction (PCR) using primers of the rolB gene. According to Southern blot analysis, one T-DNA copy was inserted into each of the hairy root lines. In the present study, transgenic hairy roots have been obtained trough genetic transformation by A. rhizogenes harbouring two plasmids, the Ri plasmid and pBI121 binary vector harbouring gus reporter gene. Expression of the gus gene in transgenic hairy root was confirmed by histochemical GUS assay.     

A Cointegrate Ti Plasmid Vector for Agrobacterium tumefaciens - mediated Transformation of indica Rice cv Pusa Basmati 1

An intermediate vector pSSJ1 was constructed by cloning a hph gene and a gus gene with catalase intron in pGV1500. pSSJ1 was cointegrated into a disarmed receptor Ti plasmid pGV2260 harboured in Agrobacterium tumefaciens strain C58C1Rif^R. The resulting A. tumefaciens strain C58C1Rif^R (pGV2260::pSSJ1) stably transformed Oryza sativa L. cv Pusa Basmati 1 scutellum-derived calli at 26% frequency. Introduction of the plasmid pSSJ3 (3′virB, virG and virC of pTiB0542) into A. tumefaciens C58C1Rif^R (pGV2260::pSSJ1) resulted in the elevation of acetosyringone-induced T -strand accumulation. Rice transformation efficiency of the cointegrate plasmid pGV2260::pSSJ1 increased from 26% to 33% in the presence of pSSJ3 and from 26% to 35% in the presence of pToK47 (complete virB, virG and virC). T-DNA integration in To plants was confirmed by Southern hybridization analysis. Inheritance analysis of the T₀ plants with single-copy T-DNA insertions revealed segregation of hygromycin resistance in 3:1 ratio. The feasibility of rice transformation with a cointegrate Ti plasmid vector is clearly established.     

T-DNA tagging of the translation initiation factor eIF-4A1 of Arabidopsis thaliana

A transgenic Arabidopsis thaliana line, generated using a T-DNA vector carrying a promoterless gus reporter gene, showed intense GUS expression in young leaves and rapidly growing stem tissues. The gus fusion has tagged the 3′ region of the gene encoding the A. thaliana eukaryotic translation initiation factor eIF-4A1. Comparison of the genomic and cDNA sequence shows that the eIF-4A1 gene contains four introns. Three introns interrupt the translated region, whereas the largest intron splits the 5′-untranslated region. In plants homozygous for the T-DNA markers, the eIF-4A1 and gus genes are expressed as different mature mRNAs. The gus gene is possibly expressed from a cryptic promoter downstream the eIF-4A1 gene.     

Induction of virulence response in Agrobacterium tumefaciens by tissue explants of various plant species

Forty-four plant species belonging to different taxa were tested for their ability to induce the expression of the virulence E gene in Agrobacterium tumefaciens containing virE:lacZ fusion constructs. With the exception of 6 algae, one fern and 2 monocots, tissue explants of all other plants (2 Algae, 3 Bryophytes, 2 Pteridophytes, 15 Gymnosperms, 8 Monocots and 5 Dicots) induced the expression of the virE gene as detected by the presence of -galactosidase activity in the bacteria.Abbreviations AS acetosyringone - vir virulence genes Scientific Contribution Number 1734 from the New Hampshire Agricultural Experiment Station     

Factors affecting genetic transformation and shoot organogenesis of Bacopa monnieri (L.) Wettst

Efficient Agrobacterium tumefaciens mediated T-DNA delivery and subsequent shoot organogenesis has been achieved from Bacopa monnieri. Various factors influenced T-DNA delivery as evident from transient GUS assay. The transient GUS expression was significantly higher (97.7 %) in explants that were pre-cultured before bacterial infection on medium supplemented with 100 μM acetosyringone. Incorporation of acetosyringone into the co-cultivation medium also enhanced transient GUS activity. Explant injury with carborundum paper, co-cultivation period of 2 days and a bacterial density of 0.4 OD₆₀₀ showed higher transient GUS expression. Following co-cultivation, shoot organogenesis was achieved from leaf segments on basal Murashige and Skoog medium containing 58 mM sucrose. Supplementation of antibiotics (cefotaxime or carbenicillin) at > 250 μg/ml into the medium significantly promoted shoot organogenesis from leaf explants (71.5 % in control and > 83.0 % on medium containing 500 μg/ml of carbenicillin or cefotaxime). Stable transformation of regenerated shoots was confirmed on the basis of GUS activity and PCR amplification of DNA fragments specific to reporter gene (uidA) and selection marker gene (nptII). The expression level of nptII gene in independent transgenic lines was studied using quantitative real time-PCR. Stable transformed shoots after rooting were successfully established in the pots.     

Rice scutellum induces Agrobacterium tumefaciens vir genes and T-strand generation

For successful transformation of a plant by Agrobacterium tumefaciens it is essential that the explant used in cocultivation has the ability to induce Agrobacterium tumour-inducing (Ti) plasmid virulence (vir) genes. Here we report a significant variation in different tissues of Indica rice (Oryza sativa L. cv. Co43) in their ability to induce Agrobacterium tumefaciens vir genes and T-strand generation, using explants preincubated in liquid Murashige and Skoog (MS) medium. An analysis of rice leaf segments revealed that they neither induced vir genes nor inhibited vir gene induction. Of different parts of rice plants of different ages analysed only scutellum from four-day old rice seedlings induced vir genes and generation of T-strands. We observed that the physical presence of preincubated scutella is required for vir gene induction. Conditioned medium from which preincubated scutella were removed did not induce the vir genes. Scutellum-derived calli, cultured for 25 days on medium containing 2,4-D, also induced virE to an appreciable level. These results suggest that scutellum and scutellum-derived calli may be the most susceptible tissues of rice for Agrobacterium-mediated transformation.     

Efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br. using shoot apices as explant source

A critical step in the development of a reproducible Agrobacterium tumefaciens mediated transformation system for a recalcitrant species, such as pearl millet, is the establishment of optimal conditions for efficient T-DNA delivery into target tissue from which plants can be regenerated. A multiple shoot regeneration system, without any intervening callus phase, was developed and used as a tissue culture system for Agrobacterium-mediated transformation. Agrobacterium super virulent strain EHA105 harboring the binary vector pCAMBIA 1301 which contains a T-DNA incorporating the hygromycin phosphotransferase (hpt II) and β-glucuronidase (GUS) genes was used to investigate and optimize T-DNA delivery into shoot apices of pearl millet. A number of factors produced significant differences in T-DNA delivery; these included optical density, inoculation duration, co-cultivation time, acetosyringone concentration in co-cultivation medium and vacuum infiltration assisted inoculation. The highest transformation frequency of 5.79% was obtained when the shoot apex explants were infected for 30 min with Agrobacterium O.D.₆₀₀ = 1.2 under a negative pressure of 0.5 × 10⁵ Pa and co-cultivated for 3 days in medium containing 400 μM acetosyringone. Histochemical GUS assay and polymerase chain reaction (PCR) analysis confirmed the presence of the GUS gene in putative transgenic plants, while stable integration of the GUS gene into the plant genome was confirmed by Southern analysis. This is the first report showing reproducible, rapid and efficient Agrobacterium-mediated transformation of shoot apices and the subsequent regeneration of transgenic plants in pearl millet. The developed protocol will facilitate the insertion of desirable genes of useful traits into pearl millet.