Tumefaciens Strain EHA105 Research Articles

Agrobacterium-mediated genetic transformation of Perilla frutescens.

A reproducible plant regeneration and an Agrobacterium tumefaciens-mediated genetic transformation protocol were developed for Perilla frutescens (perilla). The largest number of adventitious shoots were induced directly without an intervening callus phase from hypocotyl explants on MS medium supplemented with 3.0 mg/l 6-benzylaminopurine (BA). The effects of preculture and extent of cocultivation were examined by assaying beta-glucuronidase (GUS) activity in explants infected with A. tumefaciens strain EHA105 harboring the plasmid pIG121-Hm. The highest number of GUS-positive explants were obtained from hypocotyl explants cocultured for 3 days with Agrobacterium without precultivation. Transgenic perilla plants were regenerated and selected on MS basal medium supplemented with 3.0 mg/l BA, 125 mg/l kanamycin, and 500 mg/l carbenicillin. The transformants were confirmed by PCR of the neomycin phosphotransferase II gene and genomic Southern hybridization analysis of the hygromycin phosphotransferase gene. The frequency of transformation from hypocotyls was about 1.4%, and the transformants showed normal growth and sexual compatibility by producing progenies.

Agrobacterium-mediated transformation of European chestnut embryogenic cultures.

An innovative and efficient genetic transformation protocol for European chestnut is described in which embryogenic cultures are used as the target material. When somatic embryos at the globular or early-torpedo stages were cocultured for 4 days with Agrobacterium tumefaciens strain EHA105 harbouring the pUbiGUSINT plasmid containing marker genes, a transformation efficiency of 25% was recorded. Murashige and Skoog culture medium containing 150 mg/l of kanamycin was used as the selection medium. The addition of acetosyringone was detrimental to the transformation efficiency. Transformation was confirmed by a histochemical beta-glucuronidase (GUS ) assay, PCR and Southern blot analyses for the uidA (GUS) and nptII (neomycin phosphotransferase II) genes. At present, 93 GUS-positive chestnut embryogenic lines are being maintained in culture. Low germination rates (6.3%) were recorded for the transformed somatic embryos. The presence of the transferred genes in leaves and shoots derived from the germinated embryos was also verified by the GUS assay and PCR analysis.

Agrobacterium tumefaciens-mediated transformation of blueberry (Vaccinium corymbosum L.).

Transient expression studies using blueberry leaf explants and monitored by beta-glucuronidase (GUS) assays indicated Agrobacterium tumefaciens strain EHA105 was more effective than LBA4404 or GV3101; and the use of acetosyringone (AS) at 100 microM for inoculation and 6 days co-cultivation was optimum compared to 2, 4, 8, 10 or 12 days. Subsequently, explants of the cultivars Aurora, Bluecrop, Brigitta, and Legacy were inoculated with strain EHA105 containing the binary vector pBISN1 with the neomycin phosphotransferase gene (nptII) and an intron-interrupted GUS gene directed by the chimeric super promoter (Aocs)3AmasPmas. Co-cultivation was for 6 days on modified woody plant medium (WPM) plus 100 microM AS. Explants were then placed on modified WPM supplemented with 1.0 mg l(-1) thidiazuron, 0.5 mg l(-1) alpha-naphthaleneacetic, 10 mg l(-1) kanamycin (Km), and 250 mg l(-1) cefotaxime. Selection for Km-resistant shoots was carried out in the dark for 2 weeks followed by culture in the light at 30 microE m(-2) s(-1) at 25 degrees C. After 12 weeks, selected shoots that were both Km resistant and GUS positive were obtained from 15.3% of the inoculated leaf explants of cultivar Aurora. Sixty-eight independent clones derived from such shoots all tested positive by the polymerase chain reaction using a nptII primer. Eight of eight among these 68 clones tested positive by Southern hybridization using a gusA gene derived probe. The transformation protocol also yielded Km-resistant, GUS-positive shoots that were also PCR positive at frequencies of 5.0% for Bluecrop, 10.0% for Brigitta and 5.6% for Legacy.

Efficient Agrobacterium tumefaciens-mediated transformation of soybeans using an embryonic tip regeneration system

Here, we report the establishment of an efficient, in vitro, shoot organogenesis, regeneration system for soybeans [Glycine max (L.) Merr.]. Mature soybean seeds were soaked for 24 h, the embryonic tips were collected and cultured on MSB5 medium supplemented with 3.5 mg l(-1) N6-benzylaminopurine (BAP) for 24 h, and explants were transferred to MSB5 medium supplemented with 0.2 mg l(-1) BAP and 0.2 mg l(-1) indolebutyric acid. Use of embryonic tips yielded a higher regeneration frequency (87.7%) than regeneration systems using cotyledonary nodes (40.3%) and hypocotyl segments (56.4%) as starting materials. Regenerated embryonic tips were inoculated with Agrobacterium tumefaciens strain EHA105, which contains the binary vector pCAMBIA2301, and cultured for 20 h. Our results showed that the T-DNA transfer efficiency reached up to 78.2% and the transformation efficiency reached up to 15.8%. These data indicate that the embryonic tip regeneration system can be used for efficient, effective Agrobacterium-mediated transformation.

Efficient Agrobacterium tumefaciens-mediated transformation of sweet potato (Ipomoea batatas (L.) Lam.) from stem explants using a two-step kanamycin-hygromycin selection method

To achieve reliable stable transformation of sweet potato, we first developed efficient shoot regeneration for stem explants, leaf disks, and petioles of sweet potato (Ipomoea batatas (L.) Lam.) cultivar Beniazuma. The shoot regeneration protocol enabled reproducible stable transformation mediated by Agrobacterium tumefaciens strain EHA105. The binary vector pIG121Hm contains the npt II (pnos) gene for kanamycin (Km) resistance, the hpt (p35S) gene for hygromycin (Hyg) resistance, and the gusA (p35S) reporter gene for β-glucuronidase (GUS). After 3 d co-cultivation, selection of calluses from the three explant types began first with culture on 50 mg l−1 of Km for 6 wk and then transfer to 30 mg l−1 of Hyg for 6–16 wk in Linsmaier and Skoog (1965) medium (LS) also containing 6.49 μM 4-fluorophenoxyacetic acid and 250 mgl−1 cefotaxime in the dark. The selected friable calluses regenerated shoots in 4 wk on LS containing 15.13 μM abscisic acid and 2.89 μM gibberellic acid under a 16h photoperiod of 30 μmol m−2s−1. The two-step selection method led to successful recovery of transgenic shoots from stem explants at 30.8%, leaf dises 11.2%, and petioles 10.7% stable transformation efficiencies. PCR analyses of 122 GUS-positive lines revealed the expected fragment for hpt. Southern hybridization of genomic DNA from 18 independent transgenic lines detected the presence of the gusA gene. The number of integrated T-DNA copies varied from one to four.

Agrobacterium-mediated Transformation of Chokecherry (Prunus virginiana L.)

An Agrobacterium-mediated transformation system was developed for chokecherry (Prunus virginiana L.), one of the most popular native small tree or large shrub species for resource conservation and wildlife habitat in North America. Leaf tissues from in vitro plants previously maintained in MS medium with 2.5 μm BA were co-cultivated on woody plant medium (WPM) containing 10 μm BA and 200 μm acetosyringone with Agrobacterium tumefaciens strain EHA105 harboring the binary Ti plasmid pBI121 carrying the uid A gene encoding for β-glucuronidase (GUS) and the npt II gene encoding neomycin phosphotransferase II. Infected leaf explants were disinfected in sterile water and antibiotics and then transferred to WPM containing 10 μM BA and the antibiotics cefotaxime, carbenicillin, and kanamycin (CCK) for shoot regeneration at 25 °C with a 16-hour photoperiod. Agrobacterium concentration, pre-conditioning of explants, application of acetosyringone, infection time, and kanamycin tolerance of leaf tissues were evaluated for effects on transformation efficiency. Regeneration of chokecherry shoots on kanamycin-containing medium and screening by GUS histochemical assays showed that both the npt II and the uid A genes were successfully transferred into chokecherry. The transformation will be further confirmed by polymerase chain reaction (PCR) and Southern blot analyses.

Expression of the rolB gene enhances adventitious root formation in hardwood cuttings of aspen

An elite aspen hybrid (Populus × canescens × P. grandidentata) was transformed with Agrobacterium tumefaciens strain EHA105 that harbored a binary vector (pBI121) carrying the nptII gene under the nos promoter and tandem rolB-uidA (GUS) genes with the CaMV 35S or heat shock promoter. Among 32 independent kanamycin-resistant plants, 25 plants were confirmed by polymerase chain reaction and Southern blot analyses to contain all three genes, whereas five plants contained only nptII or/and uidA genes and two plants had both the rolB and nptII or uidA genes. Integration of the rolB gene significantly increased rooting ability of hardwood cuttings. Heat shock-rolB-transformed plants rooted at significantly higher percentage than the CaMV 35S-rolB-transformed plants. Heat shock treatment further enhanced rooting of heat shock-rolB-transformed plants. Exposure to exogenous auxin did not significantly increase the rooting percentage of transgenic hardwood cuttings, but increased the number of roots induced. This research shows great potential to improve rooting of hardwood cuttings of difficult-to-root woody plants which are commercially important to the horticultural and forestry industry. The transgenic plants with gain-of-function in hardwood-cutting rooting can facilitate research in the understanding of adventitious rooting from hardwood cuttings of recalcitrant woody plants.

Genetic transformation of selected mature cork oak (Quercus suber L.) trees.

A transformation system for selected mature cork oak (Quercus suber L.) trees using Agrobacterium tumefaciens has been established. Embryos obtained from recurrent proliferating embryogenic masses were inoculated with A. tumefaciens strains EHA105, LBA4404 or AGL1 harbouring the plasmid pBINUbiGUSint [carrying the neomycin phosphotransferase II (nptII) and beta-glucuronidase (uidA) genes]. The highest transformation efficiency (4%) was obtained when freshly isolated explants were inoculated with A. tumefaciens strain AGL1. Evidence of stable transgene integration was obtained by PCR for the nptII and uidA genes, Southern blotting and expression of the uidA gene. The transgenic embryos were germinated and successfully transferred to soil.

T‐DNA locus structure in a large population of soybean plants transformed using the Agrobacterium‐mediated cotyledonary‐node method

Designing transformation experiments for either functional genomics or crop improvement requires knowledge of the transgene locus structure, number, transmission and expression resulting from a specific transformation method. We recently reported an improvement to the soybean [Glycine max (L.) Merrill] cotyledonary-node transformation method that resulted in the efficient production of transgenic plants. To characterize the transgene loci resulting from this method, we analysed 270 independent T0 plants and 95 randomly selected T1 progenies for T-DNA locus complexity using Southern analysis. The lines were transformed with Agrobacterium tumefaciens strains LBA4404 or EHA105 carrying the binary plasmids pGPTV, pTOK233, pCAMBIA1303 or pCAMBIA1309, and regenerated in medium supplemented with or without silver nitrate (AgNO3). Analysis in the T0 generation showed that the number of hpt-hybridizing fragments per plant ranged from 1-15, with 31.5% of the lines having a single hpt-hybridizing fragment. Each primary soybean transformant had, on average, 2.0 unlinked transgene loci and that half of the segregating loci in the T1 progenies were single, simple T-DNA insertions. Of the loci containing multiple T-DNA fragments, a low frequency had tandem and inverted repeat T-DNA structures. Integration of binary plasmid backbone sequences occurred in 37% of primary transformants. A. tumefaciens strain, binary plasmid and thiol treatment had no significant effect on transgene locus structure, numbers or expression. Interestingly, exposure of soybean explants to AgNO3 throughout shoot induction and elongation increased T-DNA locus complexity in the primary transformants and decreased silencing of gusA expression in the T1 generation.

High-frequency transformation of Lobelia erinus L. by Agrobacterium -mediated gene transfer

A highly efficient transformation procedure was developed for Lobelia erinus. Leaf or cotyledon discs were inoculated with Agrobacterium tumefaciens strain EHA105 harboring the binary vector plasmid pIG121Hm, which contains a beta-glucuronidase gene with an intron as a reporter gene and both the neomycin phosphotransferase II and hygromycin phosphotransferase genes as selectable markers. The hygromycin-resistant calli produced on the selection medium were transferred to MS medium supplemented with 0.5 mg/l benzyladenine and 0.2 mg/l indole-3-acetic acid for regeneration of adventitious shoots. Transgenic plants were obtained as a result of the high regeneration rate of the transformed calli, which was as high as 83%. In contrast, no transgenic plant was obtained by the procedure of direct shoot formation following inoculation with A. tumefaciens. Transgenic plants flowered 3-4 months after transformation. Integration of the transgenes was detected using PCR and Southern blot analysis, which revealed that one to several copies were integrated into the genomes of the host plants. The transformation frequency at the stage of whole plants was very high--45% per inoculated disc.

Screening for highly active beta-lactam antibiotics against Agrobacterium tumefaciens.

Quantitative in vitro antibacterial activities, i.e., minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs), of 12 beta-lactam antibiotics against Agrobacterium tumefaciens strains LBA4404 and EHA101 were examined, in order to identify antibiotics effective in eliminating the bacteria in Agrobacterium-mediated plant genetic transformation. The antibacterial activities of beta-lactams tested against strain EHA101 were equal to or less than those tested against strain LBA4404. Cefotaxime, cefbuperazone, and meropenem had high activities against strain LBA4404 (MBC <1 mg l(-1)). Against strain EHA101, however, only meropenem showed activity comparable to that against strain LBA4404. The production of beta-lactamase was observed only in strain EHA101.

Agrobacterium-mediated transformation of Javanica rice cv. Rojolele.

The Agrobacterium-mediated transformation system was extended to a famous Javanica rice variety, Rojolele, that is cultivated in Indonesia now. Efficient callus induction from immature and mature seeds of Rojolele did not succeed by any previous method for any rice cultivar. In this study, the callus from mature seeds of Rojolele exhibited a compact and nodular appearance on C medium after the carbon source and medium pH was modified. Scutellum-derived calli from mature seeds were co-cultivated with Agrobacterium tumefaciens strains EHA101 or LBA4404 that carried plasmid pAFT14, which contained the genes for beta-glucuronidase (gus) and hygromycin resistance (hpt). Finally, the transformation efficiency of Rojolele variety using A. tumefaciens strain EHA101 (pAFT14) was improved to about 23%, similar to that of the Japonica rice variety Nipponbare. The seed fertility of transgenic Rojolele was more than 90%. The copy number of the transgene varied from one to three copies in the T(0) transgenic lines. Both the gus and the hpt genes were inherited and expressed in the progeny.

Transgenic regal pelargoniums that express the rolC gene from Agrobacterium rhizogenes exhibit a dwarf floral and vegetative phenotype

The regal pelargonium, ev. Dubonnet, was transformed using the disarmed Agrobacterium tumefaciens strains LBA4404 or EHA105 containing the binary vector pLN70. This plasmid carries on its T-DNA the rolC gene from Agrobacterium rhizogenes under control of the CaMV 35S promoter and the npt II selectable marker gene under a NOS promoter. Six independent transformants were produced and grouped according to their phenotypic characteristics. Two transformants showed the same phenotype as the untransformed control plants. Three transformants exhibited a dwarf phenotype and one displayed a super-dwarf phenotype. Southern hybridization analyses of the T-DNA left border region using a npt II probe showed that the six transformants all arose from independent transformation events. Northern hybridization analyses showed that the rolC gene was expressed only in the four transformants that exhibited a dwarf phenotype. Our data show that the phenotypic effects of rolC expression in regal pelargoniums include reductions in plant height, leaf area, petal area, and corolla length. Earlier flowering of the rolC transgenics by up to 22d was also observed.

Transformation of the monocotyledonous Alstroemeria by Agrobacterium tumefaciens.

An efficient procedure is described for the transformation of the monocotyledonous Alstroemeria by Agrobacterium tumefaciens via callus regeneration. Calli derived from ovules were co-cultivated with A. tumefaciens strains EHA101 and LBA4404, which harbored the binary vector plasmids pIG121Hm and pTOK233, respectively. These plasmids contain the beta-glucuronidase gene ( gusA) as a reporter gene and the hygromycin phosphotransferase and neomycin phosphotransferase II ( nptII) genes as selective markers. Inoculated calli were first plated for 4 weeks on medium containing cefotaxime to eliminate bacteria, following which time transformed cells were selected on medium that contained 20 mg/l hygromycin. A histochemical assay for GUS activity revealed that hygromycin-based selection was completed after 8 weeks. The integration of the T-DNA of pIG121Hm and pTOK233 into the genome of the cells was confirmed by PCR analysis. Efficient shoot regeneration from the transformed calli was observed on half-strength MS medium supplemented with 0.5 mg/l naphthaleneacetic acid and 0.5 mg/l benzyladenine after about 5 months of culture. The presence of the gusA and nptII genes in the genomic DNA of regenerated plants was detected by means of PCR and PCR-Southern hybridization, and the expression of these transgenes was verified by reverse transcription-PCR.

Tissue culture and Agrobacterium-mediated transformation of hemp (Cannabis sativa L.)

Hemp (Cannabis sativa L.) is cultivated in many parts of the world for ils fiber, oil, and seed. The development of new hemp cultivars with improved traits could be facilitated through the application of biotechnological strategies. The purpose of this study was to investigate the propagation of hemp in tissue culture and to establish a protocol for Agrobacterium-mediated transformation for foreign gene introduction. Stem and leaf segments from seedlings of four hemp varieties were placed on Murashige and Skoog medium with Gamborg B5 vitamins (MB) supplemented with 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 μM kinetin, 3% sucrose, and 8 gl−1 agar. Large masses of callus were produced within 4 wk for all cultivars. Suspension cultures were established in MB medium containing 2.5 μM 2,4-D. To promote embryogenesis or organogenesis, explants, callus, and suspension cultures derived from a range of explant sources and seedling ages were exposed to variations in the culture medium and changes to the culture environment. None of the treatments tested were successful in promoting plantlet regeneration. Suspension cells were transformed with Agrobacterium tumefaciens strain EHA101 carrying the binary vector pNOV3635 with a gene encoding phosphomannose isomerase (PMI). Transformed callus was selected on medium containing 1–2% mannose. A chlorophenol red assay was used to confirm that the PMI gene was expressed. Polymerase chain reaction and Southern hybridization detected the presence of the PMI gene. Copy number in different lines ranged from one to four.

Use of direct somatic organogenesis for Agrobacterium-mediated transformation of onion

An Agrobacterium tumefaciens mediated DNA delivery is being developed for onion (Allium cepa L.) using organogenic structures formed on the ovaries as target tissue. An indirect transformation procedure using A. tumefaciens strains LBA4404 and EHA105 in combination with four plasmids was used for establishing an efficient transformation protocol. Studies were focused on different protocols for co-cultivation, selection and detection of optimal reporter and selection genes in onion. A histochemical GUS test and visual detection of the GFP protein were used for optimization of transformation treatments. Several pretreatment and co­ cultivation protocols achieving transient expression were studied, and the optimal was a combination of sonication (10 s) and vacuum treatment (5min, 35 nuriHg). Among several selection media studied, optimal results were obtained using phosphinotricin (2.5 mg/1) as selection agent and timentin (150 mg/1) for good suppression of bacterial growth. The transgenic nature of individual regenerants after sixmonths on selection media was confirmed by polymerase chain reaction using primers for detection of uidA and bar genes. Using this test, three regenerants were positive for both bar and uidA genes and three regenerants were positive for the bar gene only.

조직배양을 이용한 Kentucky bluegrass(Poa pratensis L.)의 식물체 재분화 및 형질전화 조건의 검토

In this study, plant regeneration and Agrobacterium tumefaciens-mediated transformation Kentucky bluegrass(Poa pratensis L.) were evaluated. Three different types of calli were produced depending on the combinations of growth regulators. They were non-friable brown or gray-colored callus (type I), compact, friable and yellow or white-colored callus (typeII), and soft, watery translucent callus with differentiated structure (typeIII). The highest regenerable organogenic callus (typeII) was obtained on the medium containing 1mg/L, 2,4-D and 0.1mg/L BA. Additionally, the production of typeII calli increased significantly when AgNO<TEX>$_3$</TEX> was added to the callus induction and growth medium. The highest frequency of multiple shout formation from typeII callus was obtained on MS medium containing 1mg/L BA and 1mg/L Thidiazuron(TDZ). The organogenic calli(typeII) were inoculated with Agrobacterium tumefaciens strain EHA101 harboring the binary vector pIG121Hm with <TEX>$\beta$</TEX>-glucuronidase gene, and various factors were found to influence the transfer-DNA delivery efficiency. The highest transient GUS activity was observed on typeIIcallus. In the present work, we reported the first transient GUS activity of Kentucky bluegrass mediated by Agrobacterium tumefaciens. Our system may contribute to genetic improvement for breed-recalcirtrant grass species, Kentucky bluegrass.

EVALUATION OF TOLERANCE TO PIERCE'S DISEASE AND BOTRYTIS IN TRANSGENIC PLANTS OF VITIS VINIFERA L. EXPRESSING THE PEAR PGIP GENE

PG-inhibiting proteins (PGIPs) are leucine rich repeat plant cell wall proteins that specifically inhibit fungal polygalacturonases (PGs). Their role in plant defense response suggests that they may be useful for genetic engineering to obtain transgenic plants with increased tolerance to fungal infection. In addition, the fact that Xylella fastidiosa, the causal agent of Pierce’s Disease (PD) in grapevines, has genes putatively encoding PG and other cell wall-degrading enzymes led us to the hypothesis that PGIP could confer resistance against this bacterium. In order to test this hypothesis, proembryogenic calli originating from anthers of Vitis vinifera cvs. Thompson Seedless and Chardonnay were co-cultivated with Agrobacterium tumefaciens strain EHA 101 harboring binary plasmid pDU94.0928 that contains the pear pgip gene under the control of the CaMV 35S promoter. Plants from 51 independently isolated lines have been transferred to the greenhouse. Putative transgenic lines growing in the greenhouse were analyzed by PCR, Western blotting and PGIP activity assays. Western blot analysis demonstrated the presence of the protein in roots, leaves and young stems of the transgenic plants but not in untransformed controls. High levels of enzyme activity have been found in crude extracts from leaves of transgenic lines obtained from independent transformation events but not in untransformed controls. Preliminary results have shown that the development of PD in the transgenic lines analyzed so far is delayed. We also present results regarding the disease response of greenhouse-grown plants after inoculation with Botrytis cinerea.

Agrobacterium-mediated transformation of Citrus sinensis and Citrus limonia epicotyl segments

Genetic transformation allows the release of improved cultivars with desirable characteristics in a shorter period of time and therefore may be useful in citrus breeding programs. The objective of this research was to establish a protocol for genetic transformation of Valencia and Natal sweet oranges (Citrus sinensis L. Osbeck) and Rangpur lime (Citrus limonia L. Osbeck). Epicotyl segments of germinated in vitro plantlets (three weeks in darkness and two weeks in a 16-h photoperiod) were used as explants. These were co-cultivated with Agrobacterium tumefaciens strain EHA-105 and different experiments were done to evaluate the transformation efficiency: explants were co-cultivated with Agrobacterium for one, three or five days; explants were incubated with Agrobacterium suspension for 5, 10, 20 or 40 minutes; co-cultivation medium was supplemented with acetosyringone at 0, 100 or 200 µmol L-1; Explants ends had a longitudinal terminal incision (2-3 mm); co-cultivation temperatures of 19, 23 or 27°C were imposed. The experimental design was completely randomized in all experiments with five replications, each consisted of a Petri dish (100 x 15 mm) with 30 explants and resulted in a total of 150 explants per treatment. Longitudinal terminal incision in the explant ends did not improve shoot regeneration. However, transgenic plants of all three cultivars were confirmed from explants that had been subjected to inoculation time of 20 minutes, co-culture of three days at 23-27°C, in the absence of acetosyringone.

Agrobacterium-mediated transformation of Asarina procumbens Mill.

SummaryThis paper reports on protocols for plant regeneration and transformation of Asarina procumbens Mill. (syn. Antirrhinum asarina L.). Asarina is an ornamental plant, in this study we successfully achieved shoot regeneration from stem explants on MS medium supplemented with zeatin. Furthermore, the transformation system was established via Agrobacterium-mediated transformation with stem segments. The A. tumefaciens strains EHA105, GV2260 and GV3101 each harbouring binary vector pSMAK251 containing uidA and npt II genes were used in this establishment. Kanamycin-resistant shoots regenerated directly on the selection medium containing 2 mg l–1 zeatin, 100 mg l–1 kanamycin, and 250 mg l–1 cefotaxime six weeks after co-cultivation. Fifty to 73% of regenerated shoots showed GUS expression through the histochemical GUS expression analysis. PCR and Southern blot analysis confirmed the existence of npt II and uid A genes in these GUS expressed transformed plants. This is the first report of successful genetic transformation in Asarina procumbens Mill.