nptII Gene Research Articles

Immune analysis of cry1Ab-genetically modified potato by in-silico analysis and animal model.

The safety of feeding transgenic potato (Solanum tuberosum), resistant to the potato tuber moth, to Wistar rats was examined from an immunological perspective. The genetically modified potato (GMP) was harbouring cry1Ab and nptII genes as target and selectable marker genes, respectively. In-silico analysis reconfirmed that Cry1Ab and NPTII protein sequences have no significant homology to known toxins or known allergens. The Wistar rats were fed a diet containing 20% GMP or its parental control, non-GMP (NGMP), for 90days. The consumption of GMP food did not affect the growth rate, food intake, food efficiency, and general health status of the rats. There were no significant differences in the serum concentrations of immunoglobulin A (IgA), IgG, IgM, interleukin 6 (IL-6), and IFN-γ between GMP and NGMP-fed rats. Based on such data, it is concluded that the transgenic potato had no adverse effect on immunity functions of Wistar rats.

AGROBACTERIUM-MEDIATED TRANSFORMATION OF TOMATO (LYCOPERSICON ESCULENTUM Mill.) USING A SYNTHETIC Cry1Ab GENE FOR ENHANCED RESISTANCE AGAINST TUTA ABSOLUTA (Meyrick)

The effect of different plant hormones on in vitro regeneration of tomato (Lycopersicon esculentum Mill.) two cultivars Castle Rock and Super strain B, obtained from hypocotyls and cotyledons of cultured in vitro seedlings was examined. The optimal concentrations of plant growth regulators were the MS medium containing 3 mg L-1 of zeatin and 0.1 mg L-1 of NAA of cotyledon and hypocotyl explants of Super strain B cultivar. While, the best regeneration medium was the MS medium containing 1 mg L-1 ZEA, 0.5 mg L-1 BAP and 0.1 mg L-1 IAA of cotyledon and hypocotyl explants of Castle Rock cultivar. Agrobacterium‐mediated genetic transformation system has been expanded of tomato cultivar Super strain B. Transformation efficiency of hypocotyl explants was studied with Agrobacterium tumefaciens strain GV3101 harboring binary vector pICBV19, containing gus and bar genes. Transformed tomato shoots were obtained from hypocotyls explants on MS medium contained with 3 mg L-1 ZEA and 0.1 mg L-1 NAA. The gene expression of gus and bar genes was tested in the putatively transformed tomato plantlets through GUS histochemical and leaf painting assays. The plasmid pBI121 containing an insect resistance gene (cry1Ab) and kanamycin gene (nptII) as a plant selectable marker were introduced into these explants. Putatively transformed plantlets were examined by PCR and southern blot analysis. The expression of cry1Ab gene was tested through insect bioassay. For insect bioassay, experiments were conducted to determine the mortality percentage of Cry1Ab toxin protein against Tuta absoluta (Meyrick). The results indicated that Cry1Ab toxin protein expressed in transformed tomato plants showed 100% instars larval mortality was obtained after feeding for 4–5 days.

Somatic embryogenesis of Limonium sinense: a study on the regeneration efficacy via direct and indirect approach followed by Agrobacterium-mediated genetic transformation

ABSTRACTAn efficient indirect somatic embryogenesis and Agrobacterium-mediated transformation protocol for Limonium sinense has been established, wherein neomycin phosphotransferase II (npt II) and β-glucuronidase (GUS) genes were used as selectable and screenable markers, respectively. The efficiency of plantlet regeneration from transformed tissue was compared between direct embryogenesis from leaf and indirect embryogenesis from callus. Embryogenic callus (EC) was initiated from leaf explants on MS medium supplemented with 6.7 μM 2,4-D and 2.22 μM BA. The somatic embryos were induced, matured, and germinated when ECs were transferred onto MS medium supplemented with 4.44 μM BA and 1.07 μM NAA. Agrobacterium tumefaciens strain LBA 4404 containing the vector pBI121 was used for the transformation. Transient GUS expression frequency was evaluated and putative transgenic plants were successfully grown on culture medium in presence of kanamycin (80–100 mg L−1). PCR analysis of putative transgenic plants confirmed the presence of GUS and nptII genes. The transformation efficiency obtained through indirect embryogenesis from calluses (4%) was much higher than through direct embryogenesis from leaf explants (0.9%).

Inheritance and expression stability of exogenous genes in insect-resistant transgenic poplar

To understand the genetic and expression stability of transgenic insect-resistant poplar 741, this study compared the experimental plantations of transgenic insect-resistant poplar 741 lines (pb1, pb6, pb11, pb17, and pb29) with non-transgenic poplar 741, P. tomentosa Carr.f.yixianensis (poplar 84 K) and transgenic hybrid progeny lines cultured from immature embryos. The insect resistance and growth stability of transgenic poplar 741 were investigated by detecting exogenous genes by polymerase chain reaction (PCR), measuring the diameter at breast height (DBH) and volume growth, and performing insect-resistance tests against Clostera anachoreta and Hyphantria cunea. The inheritance and expression of the exogenous gene was also examined in transgenic hybrid progeny lines. The results revealed that the exogenous gene was stable, remaining stable in 8–10-year-old transgenic poplar 741 trees. No significant difference was found between the height of 10-year-old transgenic poplar 741 and non-transgenic poplar 741 in the experimental plantations in Baoding, China. The DBH and volume growth of pb17 was significantly greater than that of pb29 and pb11. The 8-year-old transgenic poplar 741 pb29 grown in Zhuozhou showed no significant difference from poplar 741 in terms of height growth, DBH, and volume. From 1999 to 2013, pb29-fed larvae (C. anachoreta larvae and H. cunea) exhibited stable mortality rates >79%. Likewise, pb11-fed larvae showed stable mortality rates (C. anachoreta larvae had mortality rates >75%, and H. cunea larvae exhibited rates >80%). pb17 conferred low insect-resistant stability, showing mortality rates that varied from 28.2 to 99.27% in C. anachoreta and H. cunea larvae. Among the hybrid progeny lines acquired by hybridization of pb1, pb29, and pb11 with 84 K poplar, the ratios of PCR-positive to PCR-negative lines for the BtCry1Ac gene were 1.31, 1.15, and 0.86, respectively. X 2 tests showed that the ratio was consistent with the Mendelian law of 1:1 segregation controlled by an allele pair. The hybrid progeny of pb6 × 84 K had a segregation ratio of 3:1. The nptII gene followed the same segregation rule as Cry1Ac. The transgenic hybrid progeny that contained Cry1Ac gene exhibited the same insect resistance as the parent plants.

Agrobacterium tumefaciens -mediated transformation of tomato (Solanum lycopersicum)

The present investigation describes the development of genetically engineered tomato plants with annexin gene. The alkaline lysis method is used to isolate the plasmid DNA of pUC 19 vector having the desired Annexin gene (3Kbp) and the plasmid DNA of the binary vector pGPTV (13 Kbp) from E.coli (DH5α strain). The purified pUC 19/Annexin and pGPTV plasmid were restriction digested using the restriction enzymes EcoRI and XbaI as a linearised band was eluted from the gel. The digested plasmid shown in the band pattern in the gel were cut by gel elution technique and purified from other reaction mixture. Then the dot spot test was done to calculate the concentration of pGPTV and Annexin gene. The recombinant PGPTV plasmid with the annexin gene in Agrobacterium tumefaciens MTCC 431 was mobilized and transferred to plant system through the mobilization helper plasmid pRK2013. The kanamycin resistance gene (NPT II) was used as a selective marker. The calli used for isolating the genomic DNA which was then amplified for confirmation of annexin gene. The nptII gene of 800 bp serves as a selectable marker system in plants and its amplification confirmed the presence of annexin gene in transgenic plants by PCR method.

Agrobacterium-Mediated Insect Resistance Gene (cry1Aa) Transfer Studies Pertaining to Antibiotic Sensitivity on Cultured Tissues of Broccoli

ABSTRACTSuccessful plant transformation requires an efficient regeneration protocol and a suitable selection method. A genetically engineered, disarmed Agrobacterium tumefaciens strain containing a binary vector pBin-1Aa with cry1Aa (insect resistance) and npt-II (neomycin phosphotransferase-II) genes was used for genetic transformation studies. Effects of antibiotics kanamycin and cefotaxime were studied to determine the aptness of kanamycin resistance as a selectable marker and for cefotaxime in controlling excessive bacterial growth during genetic transformation studies using cultured hypocotyl, cotyledon, leaf, and petiole tissues of Broccoli (Brassica oleracea L var. italica). Leaf and petiole explants exhibited decreased fresh weight as kanamycin concentration increased, resulting in full or partial inhibition of shoot regeneration. A significant or nonsignificant negative correlation occurred between kanamycin concentration and explant fresh weight over the time. Cefotaxime had little effect on regeneration potential. Growth of Agrobacterium cells was controlled at a concentration of 400 mg·L−1 cefotaxime in explants, and maximum putative transgenic shoot regeneration in explants of hypocotyls (44.88%) and cotyledons (36.29%) was obtained on MS (Murashige and Skoog)-selective shoot regeneration medium. Polymerase chain reaction analysis of genomic DNA using specifically designed primers detected the presence of the cryIAa and npt-II genes in kanamycin-resistant broccoli plantlets. Of five randomly selected putative transgenic shoots, three were positive for presence/integration of cryIAa and npt-II genes during T-DNA transfer and integration into the plant genome. Kanamycin and cefotaxime act as effective selective agents during genetic transformation pertaining to antibiotic sensitivity on cultured tissues of broccoli.

Agrobacterium tumefaciens -mediated transformation of tobacco (Nicotiana tabaccum)

The present study involves the development of genetically engineered tobacco plants with annexin gene. The plasmid DNA of pUC 19 vector having the desired Annexin gene (3Kbp) and the plasmid DNA of the binary vector pGPTV (13 Kbp) were isolated from E.coli (DH5α strain) by alkaline lysis method. The purified pUC 19/Annexin and pGPTV plasmid were restriction digested using the restriction enzymes EcoRI and XbaI as a linearised band was eluted from the gel. The digested plasmid shown in the band pattern in the gel were cut by gel elution technique and purified from other reaction mixture. Then the dot spot test was done to calculate the concentration of pGPTV and Annexin gene. The recombinant PGPTV plasmid with the annexin gene in Agrobacterium tumefaciens MTCC 431 was mobilized and transferred to plant system through the mobilization helper plasmid pRK2013. The kanamycin resistance gene (NPT II) was used as a selective marker. The calli used for isolating the genomic DNA which was then amplified for confirmation of annexin gene. The nptII gene of 800 bp serves as a selectable marker system in plants and its amplification confirmed the presence of annexin gene in transgenic plants by PCR method.

Efficient regeneration and improved sonication-assisted Agrobacterium transformation (SAAT) method for Catharanthus roseus

Catharanthus roseus is an important medicinal plant known for its pharmacological qualities such as antimicrobial, anticancerous, antifeedant, antisterility, antidiabetic activities. More than 130 bioactive compounds like vinblastine, vindoline and vincristine have been synthesized in this plant. Extensive studies have been carried out for optimization regeneration and transformation protocols. Most of the protocol described are laborious and time-consuming. Due to sophisticated protocol of regeneration and genetic transformation, the production of these bioactive molecules is less and not feasible to be commercialized worldwide. Here we have optimized the efficient protocol for regeneration and transformation to minimize the time scale and enhance the transformation frequency through Agrobacterium and sonication-assisted transformation (SAAT) method. In this study, hypocotyl explants responded best for maximal production of transformed shoots. The callus percentage were recorded 52% with 1.0 mg L−1 (BAP) and 0.5 mg L−1 (NAA) while 80% shoot percentage obtained with 4.0 mg L−1 (BAP) and 0.05 mg L−1 (NAA). The microscopic studies revealed that the expression of GFP was clearly localized in leaf tissue of the C. roseus after transformation of pRepGFP0029 construct. Consequently, transformation efficiency was revealed on the basis of GFP localization. The transformation efficiency of SAAT method was 6.0% comparable to 3.5% as conventional method. Further, PCR analysis confirmed the integration of the nptII gene in the transformed plantlets of C. roseus.

Senescence is delayed when ramie (Boehmeria nivea L.) is transformed with the isopentyl transferase (ipt) gene under control of the SAG12 promoter.

Ramie is an economically important industrial fiber crop widely planted in China, India, and other Southeast Asian and Pacific Rim countries. It plays an important role in China's economy, where ramie farming, industry, and trade provide livelihood support to about five million people. However, poor fiber production resulting from leaf senescence and leaf abscission is a significant problem. In this study, we report the successful production of transgenic ramie plants which delayed leaf senescence and enhanced biomass. Transgenic ramie plants were obtained via transformation with the Agrobacterium tumefaciens strain harboring the binary vector pSG529 containing the isopentyl transferase (ipt) gene under control of the SAG12 promoter (PSAG 12‐ipt construct). Agrobacterium tumefaciens strain EHA105 was used for the midrib explant transformation. The transformation frequency was 28.29%. Southern blot confirmed the integration of 1–4 copies of the NPTII gene into the ramie genome in the tested lines. At the fiber maturation stage, the transgenic plants had higher photosynthesis rates, chlorophyll content (SPAD values), and stronger resistance to exogenous ethylene compared with wild‐type plants.

Kanamycin selection in temporary immersion bioreactors allows visual selection of transgenic citrus shoots

In mature citrus transformation, the nptII gene is most commonly used for selection and it is confounded by the high number of non-transformed, escaped shoots that develop on semi-solid kanamycin selection medium, even at high concentrations. Selection in liquid medium with kanamycin in temporary immersion bioreactors might provide a better means of distinguishing between transformed and non-transformed shoots. A dose-response curve was constructed for wild-type Carrizo rootstock in liquid medium to evaluate the effects of kanamycin concentration on the number and the length of microshoots. Kanamycin at 200 mg/l was chosen as the optimal concentration for selection of transgenic mature citrus shoots in bioreactors. At this dose, most non-transgenic microshoots turned yellow and their lengths and numbers were significantly reduced in comparison to the no kanamycin controls. Selection of transgenic shoots in bioreactors was tested after Agrobacterium transformations of mature Carrizo and Valencia using three different binary vectors containing nptII as the selectable marker. Shoots developed on semi-solid medium and were transferred to temporary immersion bioreactors containing liquid MS medium with 200 mg/l kanamycin. After two weeks of culture in bioreactors, 21 dark green shoots were visually selected on the basis of color from a total of 6882 microshoots, and 17 of them (81%) were confirmed as transgenic with either the GUS histochemical assay, GFP fluorescence or PCR. Yellow shoots (5675) to be discarded from pTLAB21 and pCAMBIA2301 transformations were also tested for GUS or GFP expression and only one (0.01%) was positive. Kanamycin selection of mature transgenic shoots in temporary immersion bioreactors permitted transgenics to be visually distinguished on the basis of color, and reduced labor and consumable costs for PCR screening, particularly when reporter genes were not used.

Transformation of pomegranate (Punica granatum l.) a difficult-to-transform tree

Abstract In this study, an Agrobacterium-mediated transformation method was developed for pomegranate, a difficult-to-transform plant. We first optimized callus induction and shoot regeneration efficiency. Induction of calluses was best achieved from the intermodal stem sections on WPM medium supplemented with 12 μM BA and 8 μM NAA. The highest frequency of shoot regeneration (69.33%) and number of shoots (7.16) per piece of callus were obtained when calluses were incubated on WPM medium supplemented with 12 μM BA and 2 μM NAA. Callus pieces were inoculated with Agrobacterium tumefaciens strain EHA105 harboring the binary vector pBin19 carrying the neomycin phosphotransferase (nptII) gene as a selectable marker and green fluorescent protein (GFP) gene as a reporter. After 4 weeks in WPM selection medium (the best callus induction medium supplemented with 50 mg L−1 kanamycin), putative transgenic calluses were obtained. From such calluses transformed shoots began to appear approximately 3 week after the kanamycin resistance transgenic callus pieces had been placed on the best shoot regeneration medium containing 50 mg L−1 kanamycin. GFP fluorescence was observed in putatively transformed calluses and shoots. Molecular analysis confirmed the integration of the nptII transgene in transformed shoots. The transgenic shoots were rooted and successfully acclimatized.

Standardization of Agrobacterium tumefaciens-mediated genetic transformation protocol in Punica granatum L. cv. Kandhari Kabuli

A protocol for Agrobacterium tumefaciens-mediated genetic transformation in Punica granatum cv. Kandhari Kabuli has been developed using cotyledon and hypocotyl explants via both direct and indirect organogenesis pathways. Agrobacterium tumefaciens strain LBA 4404 harboring gus and npt-II genes in a binary vector pBI 121 had been used for genetic transformation studies. Pre-culturing of explants for 48 h (2 days) on regeneration medium before agrobacterial infection significantly increased the transformation frequency. Co-cultivation period of 48 h (2 days) was found to be optimum for genetic transformation. Cefotaxime in a concentration of 500 mg/l was found optimal for controlling the excessive bacterial growth and transformed shoots were selected using 50 mg/l kanamycin. Only the transformed cells were able to grow on the selective regeneration medium. Transgenic shoots were rooted on half-strength MS medium supplemented with 0.10 mg/l NAA, 0.02% activated charcoal, 50 mg/l kanamycin and 500 mg/l cefotaxime and subsequently acclimatized to the culture room conditions. Stable transformation and expression were confirmed on the basis of GUS histochemical and spectrophotometric assays. This genetic transformation protocol will provide a platform for genetic manipulation of pomegranate for incorporation of genes governing various horticulturally important traits in future.

Genetic transformation of Populus nigra X P. deltoides (black poplar, clone Gradizka)

Aim. To carry out genetic transformation of poplar Populus nigra x P. deltoides clone Gradizka with the model gene construct pCB002 carrying selective gene of kanamycin resistance and marker gene of β-glucuronidase. Methods. Genetic transformation was performed with the using leaf, stem and petiole poplar explants. Transformants were selected on the medium with kanamycin, and transgene was identified by polymerase chain reaction (PCR) and histochemical GUS assay. Results. Successful transformants selected on kanamycin media were confirmed by the presence of PCR-product for the gene nptII with the length 700 bp, and gus gene expression was also observed. Conclusions. Protocol for genetic transformation of P. nigra x P. deltoides clone Gradizka established here will be used for poplar genetic modification to create new clones with commercially important traits. Keywords: genetic transformation, Populus sp., microclonal propagation.

Genetic transformation of gentian Gentiana tibetica (King) leaf explants with Agrobacterium tumefaciens strain C58C1

Experiments were carried out on transformation of leaf explants derived from the axenic culture of Gentiana tibetica (King) using a co-culture of Agrobacterium tumefaciens. A. tumefaciens octopine strain C58C1 carried neomycine phosphotransferase (nptII) and β-glucuronidase (uidA) genes. The influence of plasmid helper pCH32 on transformation efficiency was stressed. After co-cultivation, explant was cultured on three different regeneration media (RM1-3) at the presence of the timentin and callus formation and plants regeneration occurred. The transgenic character of the selected tissue and transformants T0 in the presence of kanamycin has been confirmed by the histo-chemical analysis of reporter enzyme activity (ß-glucuronidase), polymerase chain reaction, and southern hybridization detecting uidA and nptII genes. l-glutamine used during inoculation period had a significant influence on the transformation efficiency in relation to its concentration and time of treatment.

Studies on Trans-Generational Transcriptional Silencing of <i>cry</i>1<i>Ac</i> Gene in Tobacco Transgenics

Developing transgenics that express high levels of Cry1Ac protein, and at the same time, are phenotypically normal, has not been an easy task to achieve. It has been routinely observed that most of the transgenic plants that survive, show no or extremely low levels of Cry1Ac protein. However, all of these plants do express the selectable marker, nptII gene. In the present study, we record an interesting observation of how one of the genes (cry1Ac) on a single T-DNA fragment is selectively silenced, keeping the expression of the other gene (nptII) intact. Further, this silenced state is inherited.

Establishment of an efficient Agrobacterium-mediated genetic transformation system in Pelargonium graveolens: an important aromatic plant

Rose-scented geranium is an important aromatic herb, have eminent for oil. The oil of geranium commercially utilized in the perfumery, cosmetic and aromatherapy industries all over the world. It is also helpful to cure many of the diseases, since it possess antibacterial, antifungal, antioxidant, anti-inflammatory and anticancer activities. However rose scented geranium suffer from several biotic and abiotic stresses, which reduced the yield of oil. So there is need to genetically improve the geranium using biotechnological approaches. The present study demonstrates the establishment of direct regeneration and Agrobacterium tumefaciens (LBA4404) mediated transformation protocol in Pelargonium graveolens (cv. CIM-BIO 171). Different media combinations such as benzyl amino purine (BAP), kinetin, naphthalene acetic acid (NAA), and adenine di-sulphate (ADS) were standardised to induce direct regeneration in P. graveolens. The maximum regeneration frequency i.e. 90.56 ± 1.2% per explant was achieved from petiolar segments in medium containing 2.5 mg/l BAP, 0.1 mg/l NAA, 1 mg/l ADS. However, with the leaf explants only 45.94 ± 2.91% frequency was achieved. In the present study, A. tumefaciens strain LBA4404 was used carrying binary vector pBI121 with the gusA as a reporter gene and neomycin phosphotransferase II (nptII) gene as a plant selectable marker. Parameters like bacterial optical density, infection time, acetosyringone concentration and kanamycin concentration were optimised to achieve maximum transformation frequency (69.5 ± 2.3%).The putative transgenic shoots were subsequently rooted on half strength MS medium and successfully transferred to the greenhouse. The transgenic plants were characterised by gus histochemical assay, PCR analysis (nptII-786 bp and gus A- 1707 bp) and Southern hybridization tests using gusA gene probe. The regeneration as well as transformation protocol will no doubt provide the basis to decipher the insights of metabolic pathways in geranium. Also could be useful for genetic improvement, to make it more tolerant/resistant against biotic and abiotic stresses and ultimately fruitful for Indian farmers in agronomic traits like high biomass, oil content, yield and better quality.

An efficient protocol for transferring selectable marker gene gus/gusplus into cassava plants (Manihot esculenta Crantz) mediated by bacteria Agrobacterium tumefaciens

In this study, to evaluate the ability to accept transgenes of the two cassava cultivars KM94 and KM140, which are grown widely in Viet Nam, A. tumefaciens bacterial strains C58/pGV2260, EHA105 and LBA4404 containing vector pCB-gusplus or pPIPRA558 haboring selectable marker gene gus/gusplus, were co-inoculated with explants from four selected sources, including (1) immature leaves, (2) shoot apexes, (3) callus, and (4) somatic embryo cotyledons. Transgenic explants were selected using three antibiotics kanamycin, neomycin, and paromomycin, at concentrations of 25, 50, 75, and 100 mg/l , based on nptII gene. These experiments were conducted to optimize conditions for transferring gus gene to cassava plant. The transformation efficiency was evaluated based on the percentage of X-gluc positive stained explants 10 days after infection, somatic embryos, regenerated shoots and whole regenerated plants. The highest transformation efficiency was achieved when using A. tumefaciens C58/pGV2260, carrying expression vector pCB-gusplus, and cotyledons of cultivars KM94. In this protocol, cotyledons were cut into small pieces, then cultured on the callus induction medium for 2 days and submerged in bacterial suspension, supplemented with 100 μM AS, with shaking at 50 rpm for 15 minutes. Explants were then co-cultured on somatic embryo induction medium supplemented with 150 μM AS in the darkness for 2 days. Explants were then transferred to selective callus induction medium with 50 mg/l kanamycin for 3 - 4 weeks, followed by the culture on selective shoot induction medium with the same kanamycin concentration. Shoots, with 2 - 3 leaves, were transferred to a selective rooting medium to establish whole plants.

Genetic transformation of pigeonpea (Cajanus cajan L.) and screening transgenic progenies based on lateral root inhibition

Production of transgenic pigeonpea is becoming increasingly important, but the methods currently employed in production and subsequent screening still requires improvement. Here, we describe Agrobacterium-mediated genetic transformation of pigeonpea with reporter uidA (gus) gene and selectable marker, neomycin phospho-transferase (nptII) gene. Histochemical assay demonstrate localization of gus activity in cells and transformed plants. Overall, a transformation frequency of 0.33% was achieved using the protocol. Grafting of in vitro-regenerated healthy shoots indicates higher survival percent (72.6%), when stock and scion are of the same variety. Seeds harvested from primary transgenic plants can be screened based on lateral root inhibition strategy. Approximately 87% of the screened T1 plants were found to be PCR positive. In conclusion, in vitro grafting of transgenic pigeonpea shoots leads to better plant establishment and screening based on lateral root inhibition leads to quick identification of positive segregants.

Optimization of Agrobacterium-mediated genetic transformation of shoot tip explants of green gram (Vigna radiata (L.) Wilczek)

Shoot tip explants prepared from seedlings of ML-267 genotype of green gram were inoculated on MSB5 medium supplemented with BAP (0–20 μM) individually or in combination with minimal concentration of auxins (NAA/IAA/IBA) for adventitious shoots formation. BAP alone without auxins was observed to be efficient in multiple shoot induction and optimum shoot proliferation was achieved on MSB5 medium containing 10 μM BAP with 100 % shoot induction frequency. 3-day-old explants gave best shoot multiplication response and the mean shoot number decreased significantly in 4-day and 5-day-old explants. The induced shoots rooted profusely on ½ MSB5 + 2.46 µM IBA and about 90 % of the plantlets survived after acclimatization and set seed normally. Shoot tip explants infected with A.tumefaciens (LBA4404) harboring pCAMBIA 2301 + AnnBj1 recombinant vector. Various factors which influence the competence of transformation were optimized based on the frequency of transient GUS expression in shoot tip explants. Optimum levels of transient GUS expression were recorded at pre-culture of explants for 2 days, infection for 10 min with Agro-culture of 0.8 OD and co-cultivation for 3 days on co-cultivation medium containing 100 µM acetosyringone in dark at 23 °C. Putative transformed shoots were produced on selection medium (shoot inductionmedium with100 mg/l kanamycin and 250 mg/l cefotaxim). PCR analysis confirmed the presence of AnnBj1, nptII, and uidA genes in T0 plants. Stable GUS activity was detected in flowers of T0 plants and leaves of T1 plants. PCR analysis of T1 progeny revealed AnnBj1 gene segregated following a Mendelian segregation pattern.

35S Promoter Methylation in Kanamycin-Resistant Kalanchoe (Kalanchoe pinnata L.) Plants Expressing the Antimicrobial Peptide Cecropin P1 Transgene.

Transgenic kalanchoe plants (Kalanchoe pinnata L.) expressing the antimicrobial peptide cecropin P1 gene (cecP1) under the control of the 35S cauliflower mosaic virus 35S RNA promoter and the selective neomycin phosphotransferase II (nptII) gene under the control of the nopaline synthase gene promoter were studied. The 35S promoter methylation and the cecropin P1 biosynthesis levels were compared in plants growing on media with and without kanamycin. The low level of active 35S promoter methylation further decreases upon cultivation on kanamycin-containing medium, while cecropin P1 synthesis increases.