Agrobacterium tumefaciens-mediated transformation of Pisum sativum L. using binary and cointegrate vectors

Epicotyl segments and nodus expiants from etiolated seedlings of Pisum sativum were transformed using Agrobacterium tumefaciens strains GV 2260 (p35S GUS INT) and GV 3850 HPT carrying either a neomycin- or hygromycinphosphotransferase-gene as selectable markers. The transgenic character of hygromycin- or kananamycin-resistant tissue was confirmed by detection of nopaline or neomycinphosphotransferase-II- and ß-glucuronidase activity in crude extracts of resistant tissues. Up to 5 % of developing shoots from shoot proliferating nodi were regenerated via organogenesis to kanamycin-resistant plantlets. Transformation frequency in vitro was found to be influenced by expiant source, A. tumefaciens strain, pea genotype and duration of cocultivation. Acetosyringone did not increase the transformation rate.Abbrevations GUS ß-glucuronidase - NAA 1-naphthyl-acetic-acid - BA 6-benzylaminopurine - NPT-II neomycinphosphotransferase-II - HPT hygromycinphosphotransferase     

Efficient shoot regeneration of Brassica campestris using cotyledon explants cultured in vitro

Summary A system has been developed for efficient regeneration of shoots from Brassica campestris in vitro. Using 4-day old cotyledons with petioles as expiants and a combination of BA and NAA in the regeneration media, up to 70% of expiants produced shoots after 2 weeks in culture. The optimal conditions for regeneration were found to include a BA concentration of 2mgL^–1 and NAA concentration of 1mgL^–1. Light intensity had a profound effect on regeneration potential. The use of silver ions as an inhibitor of ethylene action reduced regeneration rates in this system. Rooting occured simultaneously with shoot formation on these media and the resultant shoots could be rooted readily on minimal medium. The genotype dependency was investigated and indicated that this method would be widely applicable to B. campestris cultivars. Regeneration of one cultivar, a high erucic acid type (R-500), was inefficient in the system described here. Histological studies indicated the development of multiple shoot primordia from the petiolar cut ends of the expiants after the initiation of meristematic activity in the cells about 100m from the cut site within 2 days of culture initiation. The system described is compatible with previously reported Agrobacterium — mediated transformation protocols involving cotyledonary petioles.     

Efficient transformation of Arabidopsis thaliana: comparison of the efficiencies with various organs,plant ecotypes and Agrobacterium strains

Summary The efficiency of Agrobacterium-mediated transformation of Arabidopsis thaliana was compared with different organs, Arabidopsis ecotypes, and Agrobacterium strains. Efficiency of shoot regeneration was examined using hypocotyl, cotyledon and root explants prepared from young seedlings. Hypocotyl expiants had the highest regeneration efficiency in all of the four Arabidopsis ecotypes tested, when based on a tissue culture system of callus-inducing medium (CIM: Valvekens et al. 1988) and shoot-inducing medium (SIM: Feldmann and Marks 1986). Histochemical analysis using the ß-glucuronidase (GUS) reporter gene showed that the gusA gene expression increased as the period of preincubation on CIM was extended, suggesting that dividing cells are susceptible to Agrobacterium infection. In order to obtain transgenic shoots, hypocotyl explants preincubated for 7 or 8 days on CIM were infected with Agrobacterium containing a binary vector which carries two drug-resistant genes as selection markers, and transferred to SIM for selection of transformed shoots. Of four Arabidopsis ecotypes and of three Agrobacterium strains examined, Wassilewskija ecotype and EHA101 strain showed the highest efficiency of regeneration of transformed shoots. By combining the most efficient factors of preincubation period, Arabidopsis ecotype, tissue, and bacterial strain, we obtained a transformation efficiency of about 80–90%. Southern analysis of 124 transgenic plants showed that 44% had one copy of inserted T-DNA while the others had more than one copy.Abbreviations AIM Agrobacterium infection medium - CIM callus-inducing medium - CTAB cetyltrimethylammonium bromide - 2,4-D 2,4-dichlorophenoxy-acetic acid - GUS ß-glucuronidase - hph hygromycin phosphotransferase - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2ip N -(2-isopentenyl) adenine - NPTII neomycin phosphotransferase II - RIM root-inducing medium - 35S cauliflower mosaic virus 35S promoter - SIM shoot-inducing medium     

Rapidin Vitro propagation and enhanced stevioside accumulation inStevia rebaudiana Bert

A very rapid and efficient regeneration method has been established using mature expiants ofStevia rebaudiana Bert. Adventitious shoots were induced from nodal expiants of field-grown plants on four basal media supplemented with various combinations of auxins and cytokinins. The best performance (23.4 ± 2.1 shoots per expiant) was obtained on a Murashige and Skoog (MS) medium containing 2 mg L^-1 IAA and 0.5 mg L^-1 kinetin. Roots were then produced when thesein vitro- regenerated shoots were transferred to an MS medium supplemented with 3% sucrose and 2 mg L^-1 IBA. When acclimatized to soil, the rooted plants had a 98.4% survival rate. Following transplantation in the field, stevioside contents were similar between the regenerated plants (10.68 mg g^-1 dry weight) and the mother plants (12.01 mg g^-1 dw).     

Histology and chimeral segregation reveal cell-specific differences in the competence for shoot regeneration and Agrobacterium-mediated transformation in Kohleria internode explants

Summary Internode explants of Kohleria sp. (Gesneriaceae) are capable of regenerating large numbers of adventitious shoots. Regeneration of green shoots from explants of an albino periclinal chimera with genetically green L1, as well as microsurgical removal of the epidermis revealed that shoots originate only from the epidermis. Histological studies further showed that shoots arise from a particular epidermal cell type, viz the basal cell of young glandular trichomes. On the other hand, cells competent for Agrobacterium-mediated transformation are mainly located in vascular tissues, as could be shown by histochemical localization of ß-glucuronidase (GUS) expression in explants that had been inoculated with A. tumefaciens strains carrying binary plasmids with GUS and kanamycin resistance (NPTII) genes. Only 3% of GUS expression events took place in the epidermis. Consequently, shoot regeneration in the presence of kanamycin was very poor. Moreover, most of those shoots proved GUS-negative and did not survive subcultivation on kanamycin-containing medium. Six regenerants, however, were most probably transgenic, as suggested by the ability to produce adventitious shoots in the presence of kanamycin and by polymerase chain reaction (PCR) analysis. To our knowledge, this is the first positive result towards genetic transformation in a taxon of the Gesneriaceae.Abbreviations BA N⁶-benzyladenine - ct cefotaxime - GUS ß-glucuronidase - IAA indole-3-acetic acid - km kanamycin - NPTII neomycin phosphotransferase II - PCR polymerase chain reaction     

High efficiency Agrobacterium-mediated gene transfer to flax

Summary Using an Agrobacterium tumefaciens binary vector (pAL4404, pBI131), we have demonstrated the transfer of the -glucuronidase gene into the flax (Linum usitatissimum L.) cultivar Glenelg after selection for kanamycin resistance. The transformed lines were obtained by inoculation and subsequent regeneration of hypocotyl segments. The callus that formed on the cut surfaces of the hypocotyl segments was isolated three weeks after infection and was subsequently subcultured to yield shoots. This procedure generated a large number of transgenic shoots over a relatively short period of time. The transformation efficiencies obtained were the highest reported so far for this plant species.Abbreviations 2,4-D, 2,4 dichlorophenoxyacetic acid - GUS glucuronidase - MS Murasbige and Skoog (1962) medium - MU 4-methyl-umbelliferone - MUG 4-methylumbelliferyl-glucuronide - NPTII neomycin phosphotransferase II - PCR polymerase chain reaction     

Transformation of sunflower (Helianthus annuus L.): a reliable protocol

Summary A reliable protocol for the transformation of cultivated sunflower (Helianthus annuus L.) has been established, based on microprojectile bombardment of half shoot apices in combination with Agrobacterium tumefaciens coculture. Transgenic shoots have been obtained from 5 inbred lines, although transformation efficiencies varied with the genotype. Plants expressing the transgenes could be recovered from up to 7% of the explants. A minority of plants was shown to be chimaeric for expression of ß-glucuronidase activity while most appeared to be uniformly transformed. Genetic segregation was 31 for both ß-glucuronidase and neomycine phospho transferase in some plants, indicating that the respective mother plants were uniformly transformed. Integration of the foreign genes was also shown by Southern analysis.Abbreviations BAP benzyl amino purine - EDTA ethylene diamine tetraacetic acid - GUS ß-glucuronidase - npt II neomycine phospho-transferase II     

Optimisation of DNA transfer and transientβ-glucuronidase expression in electroporated maize (Zea mays L.) microspores

Summary The ability to deliver free DNA into microspores of a highly androgenic hybrid of maize was assessed by electroporation, using a square wave pulse discharge apparatus. The electroporation medium was chosen according to its ability to maintain a high level of regeneration. Nuclease activities were analyzed and were inhibited by the addition of 100 mM KNO₃ and MgSO₄ in the electroporation medium. Seven expression vectors withUid A as the reporter gene under the control of cauliflower mosaic virus 35S, Lat 52-7, Zmg 13, Emu, Ubiq-1, Al, or Actl promoters were tested in relation to the level of ß-glucuronidase expression in maize microspores. The highest level of expression was obtained when theUid A gene was driven by the Actl promoter. Therefore, this vector was further used to define optimal conditions leading to highest levels of ß-glucuronidase expression. The parameters determined in this study could provide an ideal starting point for the obtention of transgenic maize plants from electroporated microspores.Abbreviations DH diplohaploid - PEG polyethylene glycol - GUS ß-glucuronidase - EDTA Ethylene-diaminetetra Acetic acid - CaMV Cauliflower mosaic virus     

Far upstream activating promoter regions are responsible for expression of the BnC1 cruciferin gene from Brassica napus

Summary Cruciferin is the major seed storage protein in Brassica napus. As much as 1.9 kbp of the BnC1 cruciferin gene promoter have been sequenced and analyzed. Promoter fragments with 5 deletions from –2500 to –v202 were fused with the ß-glucuronidase reporter gene and used for Nicotiana tabacum transformation. ß-glucuronidase could be specifically expressed in transgenic tobacco seeds under the control of the BnC1 promoter and regulatory elements were found to be dispersed over 1903 bp. An almost 5-fold increase in ß-glucuronidase expression was obtained when the promoter length was increased from –379 to –498, and another 10-fold increase was observed when sequences between –1266 and –1903 were added. Histochemical analysis shows that the region between –844 and –1266 directs the expression of the chimeric gene specifically to the root apical meristem.Abbreviations GUS ß-glucuronidase - MU 4-methyl umbelliferone - MUG 4-methyl-umbelliferyl-ß-D-glucuronide - X-gluc 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide     

Agrobacterium-mediated transformation of almond leaf pieces

A protocol for Agrobacterium-mediated transformation of leaf pieces from two almond cultivars has been developed. This protocol gave a transformation percentage of 48.6 for the cultivar MN51 and 27.9 for the cultivar Supernova. The Agrobacterium tumefaciens strain LBA 4404 carrying a binary vector plasmid pBinGUSint has been used. A critical step for optimal transformation was the pre-culture period of leaf pieces prior selection on medium containing kanamycin. Both cultivars showed an almost doubled transformation percentage when the selection was started at day 6 compared to day 0. The presence of neomycin phosphotransferase II and ß-glucuronidase genes carried by pBinGUSint was confirmed in transformed expiants both by Southern blotting analysis and enzyme activity assays.Abbreviations CaMV cauliflower mosaic virus - GUS ß-glucuronidase - NPTII neomycin phosphotransferase II - -NAA -naphthaleneacetic acid - BAP benzylaminopurine - IAA indole-3-acetic acid - Kin kinetin - kn kanamycin - cx cefotaxime - cb carbenicillin - Tris trishydroxymethylaminomethane - PVPP insoluble polyvinylpyrrolidone-40 - X-gluc 5-bromo-4-chloro-3-indolyl-ß-D-glucuronic acid - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecil sulfate - SSC sodium chloride (3 M) sodium citrate (0.3 M)     

Agrobacterium-mediated transformation of apple (Malus x domestica Borkh.): an assessment of factors affecting regeneration of transgenic plants

We have previously developed a protocol for efficient gene transfer and regeneration of transgenic calli following cocultivation of apple (cv. Jonagold) explants with Agrobacterium tumefaciens (De Bondt et al. 1994, Plant Cell Reports 13: 587–593). Now we report on the optimization of postcultivation conditions for efficient and reproducible regeneration of transgenic shoots from the apple cultivar Jonagold. Factors which were found to be essential for efficient shoot regeneration were the use of gelrite as a gelling agent and the use of the cytokinin-mimicing thidiazuron in the selective postcultivation medium. Improved transformation efficiencies were obtained by combining the hormones thidiazuron and zeatin and by using leaf explants from in vitro grown shoots not older than 4 weeks after multiplication. Attempts to use phosphinothricin acetyl transferase as a selectable marker were not successful. Using selection on kanamycin under optimal postcultivation conditions, about 2% of the leaf explants developed transgenic shoots or shoot clusters. The presence and expression of the transferred genes was verified by -glucuronidase assays and Southern analysis. The transformation procedure has also been succesfully applied to several other apple cultivars.Abbreviations BAP benzylaminopurine - CTAB hexadecyltrimethylammoniumbromide - Na₂EDTA ethylenediamine-tetra-acetate ferric-sodium salt - FeNaEDTA ethylenediamine-tetra-acetate ferric-sodium salt - GA₃ gibberellic acid 3 - GusA -glucuronidase - gusA -glucuronidase gene of Escherichia coli - IAA indole acetic acid - IBA indole butyric acid - 2iP N6-2-isopentenyl adenine - NAA naphthalene acetic acid - nptII neomycinphosphotransferase II gene - bar phosphinothricin acetyl transferase gene - PCR polymerase chain reaction - PPT phosphinothricin - STS silver thiosulphate - T-DNA transferred DNA - TDZ thidiazuron - X-Gluc 5-bromo-4-chloro-3-indolyl -D-glucuronide - Zea trans-Zeatin     

Agrobacterium-mediated transformation of sweet orange and regeneration of transgenic plants

Summary Transgenic sweet orange (Citrus sinensis L. Osbeck) plants have been obtained by Agrobacterium tumefaciens-mediated gene transfer. An hypervirulent A. tumefaciens strain harboring a binary vector that contains the chimeric neomycin phosphotransferase II (NPT II) and ß-glucuronidase (GUS) genes was cocultivated with stem segments from in vivo grown seedlings. Shoots regenerated under kanamycin selection were harvested from the stem segments within 12 weeks. Shoot basal portions were assayed for GUS activity and the remaining portions were shoot tip grafted in vitro for production of plants. Integration of the GUS gene was confirmed by Southern analysis. This transformation procedure showed the highest transgenic plant production efficiency reported for Citrus.Abbreviations BA benzyladenine - CaMV cauliflowermosaic virus - GUS ß-glucuronidase - LB Luria Broth - MS Murashige and Skoog - NAA naphthalenacetic acid - NOS nopaline synthase - NPT II neomycin phosphotransferase II - PEG polyethylene glycol - RM rooting medium - SRM shoot regeneration medium     

Transient gene expression in strawberry (Fragaria x ananassa Duch.) protoplasts and the recovery of transgenic plants

Summary A transient ß-glucuronidase (GUS)-assay was performed to evaluate electroporation parameters and optimize DNA delivery conditions into strawberry protoplasts. Optimal GUS-activity was obtained when protoplasts were subjected to 400 V/cm for 20 ms. GUS-activity could be further increased by the addition of carrier DNA to the electroporation mixture. Callus selected on 10 g/ml hygromycin produced shoots which exhibited GUS-activity. The transformed nature of the shoots obtained after selection was confirmed by DNA-analysis.Abbreviations CaMV cauliflower mosaic virus - dCTP deoxycytidine-triphosphate - EtBr ethidium bromide - GUS ß-glucuronidase - MES 2(N-morpholino) -ethanesulfonic acid - X-gluc 5-bromo-4-chloro-3-indolyl glucuronide     

Factors that affect plant regeneration from in vitro culture of immature seeds in four lentil cultivars

An efficient and simple method for plant regeneration from immature lentil seeds (Lens culinaris) is described. Immature seeds from 1 to 6 mm of four lentil cultivars were cultured in vitro on 10 different media. Culture media included different concentrations of N ⁶ -benzylaminopurine (BAP), alone or in combination with other phytohormones. After 4 weeks in culture, multiple shoot regeneration was observed using media with BAP. Immature seed size showed significant effect on shoot regeneration. Regenerated shoots (up to 4 shoots per explant on medium with Kinetin (KN) and from 5 to 20 on media with BAP) formed adventitious roots 30 days after transferring them to a medium containing 11.4 μM indole-3-acetic acid (IAA). The efficiency of the rooting medium varied depending upon the shoot-regeneration medium and the cultivar tested. The highest rooting percentage (88.9%) was obtained from regenerated shoots of the cultivar Verdina on a medium with 1 μM α-naphthaleneacetic acid (NAA). This revised version was published online in June 2006 with corrections to the Cover Date.     

Agrobacterium-mediated transformation of the desiccation-tolerant plant Craterostigma plantagineum

Summary An efficient procedure for Agrobacterium tumefaciens- mediated transformation of the desiccation-tolerant plant Craterostigma plantagineum has been developed. Leaf explants were inoculated with A. tumefaciens strain GV3101 carrying the gene for kanamycin- or hygromycin-resistance and the ßglucuronidase reporter gene. Parameters which affected the transformation efficiency were the age of the explant, the degree of wounding and the presence of an antioxidant in the medium. Under optimal conditions, calli originated in more than 80% of leaf explants. Transformed plants were obtained from more than 50% of the cultured calli during regeneration in the presence of a suitable antibiotic. The stable integration of T-DNA was confirmed by Southern blot analysis and its expression by assays for ß-glucuronidase activity.Abbreviations GUS ß-glucuronidase - MUG 4-methyl-umbelliferyl ß-D-glucuronide - ABA abscisic acid - NPTII neomycin phosphotransferase II - CaMV cauliflower mosaic virus - MSAR modified MS medium - MS Murashige and Skoog     

Plant regeneration and transient GUS expression in a range of lavandin (Lavandula × intermedia Emeric ex Loiseleur) cultivars

Five cultivars of lavandin were compared for their ability to regenerate plantlets in vitro and for their susceptibility to genetic transformation. Both processes were shown to be strongly cultivar-dependent. For regeneration, best results were obtained with cultivar ‘37–70’ which gave an average of 7 shoots from one initial explant after 4 months culture in vitro. The other cultivars produced between 0.5 and 3.5 shoots per explant. These differences were mostly due to the variable efficiency of the shoot elongation and rooting steps. An Agrobacterium-mediated transformation system using the β-glucuronidase and neomycin phosphotransferase II genes was established. The β-glucuronidase expression was analysed for both leaf explants six days after inoculation and kanamycin-resistant calluses obtained after a six-week culture on a selective medium. For each cultivar, kanamycin-resistant calluses showing a β-glucuronidase activity were obtained. The transformation efficiency ranged from 3% for cultivar ‘Certitude’ to 89% for cv. ‘41–70’ and ‘B–110’. Some kanamycin-resistant calluses were organogenic. This revised version was published online in June 2006 with corrections to the Cover Date.     

Direct shoot regeneration from nodal,internodal and petiolar segments of <Emphasis Type="Italic">Piper longum</Emphasis> L. and in vitro conservation of indexed plantlets

Three explants namely, nodal, internodal and petiolar segments were used to establish in vitro cultures of Piper longum. Multiple shoots were induced on semi-solid Murashige and Skoog (MS) medium supplemented with 1 mg/l 6-benzyladenine (BA). Addition of ascorbic acid (40 mg/l) considerably reduced browning of tissue and medium. Best shoot regeneration was observed from petiolar explants and was, therefore, used for all further studies. An indexing method was introduced for checking bacterial contamination in well established shoot multiplication cultures. It was found that bacterial infection was quite high in shoots derived from nodal and internodal explants while it was least in those obtained from petiolar segments. Only shoots that indexed negative for endogenous bacteria were used for proliferation and in vitro conservation studies. At the end of 4 weeks in proliferation medium which consisted of MS supplemented with 0.5 mg/l BA and 40 mg/l ascorbic acid as many as 22 shoot buds of 41 mm length could be obtained. Shoot buds developed into clusters for ease of further proliferation. A step of shoot elongation for 2 weeks in liquid MS basal medium was found to be beneficial for getting long and healthy shoots for rooting. Single shoots were rooted in 0.25 mg/l indole butyric acid that could be successfully acclimatized under nethouse conditions. A conservation strategy was also developed. The shoot cultures could be maintained without subculturing for as long as 8 weeks in MS medium supplemented with 1 mg/l paclobutrazol (PBZ) and 40 mg/l ascorbic acid.     

Optimization of polyethylene glycol mediated transient gene expression in pea protoplasts

We have investigated factors influencing polyethylene glycol mediated DNA uptake and ß-glucuronidase expression in pea (Pisum sativum L.) protoplasts. It was found that for optimal \-glucuronidase expression the molecular weight and concentration of polyethylene glycol should be 4000 and 20%, respectively. The amount of plasmid DNA should be 25 g per 5×10⁵ protoplasts in each treatment, and the concentration of Mg²⁺ in the transformation buffer should be 15 mM. The optimized protocol was applicable to all four pea cultivars tested.Abbreviations FDA fluorescein diacetate - GUS ß-glucuronidase - MU 4-methylumbelliferone - MUG 4-methyl umbelliferyl glucuronide - MW molecular weight - PEG polyethylene glycol - X-Gluc 5-bromo-4-chloro-3-indolyl glucuronide     

The transformation of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.): applicable methods of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated gene transfer

Three methods of transformation of pea (Pisum sativum ssp. sativum L. var. medullare) were tested. The most efficient Agrobacterium tumefaciens-mediated T-DNA transfer was obtained using embryonic segments from mature pea seeds as initial explants. The transformation procedure was based on the transfer of the T-DNA region with the reporter gene uidA and selection gene bar. The expression of β-glucuronidase (GUS) in the regenerated shoots was tested using the histochemical method and the shoots were selected on a medium containing phosphinothricin (PPT). The shoots of putative transformants were rooted and transferred to non-sterile conditions. Transient expression of the uidA gene in the tissues after co-cultivation and in the course of short-term shoot cultivation (confirmed by histochemical analysis of GUS and by RT-PCR of mRNA) was achieved; however, we have not yet succeeded in proving stable incorporation of the transgene in the analysed plants.     

Organogenesis in vitro of Nothofagus alpina (P. et E.) Oerst, Fagaceae

In vitro shoot and root regeneration of 2-year-old Nothofagus alpina plants was achieved from several types of expiants cultured in vitro on a modified Woody Plant Medium formulation. Multiple shoot formation was obtained from leaf expiants using 0.45–2.27 M thidiazuron and 0.0049–0.098 M indolebutyric acid. Excised axillary buds formed shoots and roots in the presence of 0.0049 M benzyladenine and 2.46 M indolebutyric acid, or in the absence of plant growth regulators. Nodal sections rooted when 2.46 M indolebutyric acid at was supplied in the medium. Subcultured shoots originating from nodal sections showed a high regeneration rate through multiple shoot and root formation.Abbreviations BA benzyladenine - GA₃ giberellic acid - IBA indolebutyric acid - NAA -naphthaleneacetic acid - TDZ thidiazuron - WPM McCown's Woody Plant Medium - Z zeatin