Improved methods in Agrobacterium–mediated transformation of almond using positive (mannose/pmi) or negative (kanamycin resistance) selection-based protocols

A protocol for Agrobacterium-mediated transformation with either kanamycin or mannose selection was developed for leaf explants of the cultivar Prunus dulcis cv. Ne Plus Ultra. Regenerating shoots were selected on medium containing 15 μM kanamycin (negative selection), while in the positive selection strategy, shoots were selected on 2.5 g/l mannose supplemented with 15 g/l sucrose. Transformation efficiencies based on PCR analysis of individual putative transformed shoots from independent lines relative to the initial numbers of leaf explants tested were 5.6% for kanamycin/nptII and 6.8% for mannose/pmi selection, respectively. Southern blot analysis on six randomly chosen PCR-positive shoots confirmed the presence of the nptII transgene in each, and five randomly chosen lines identified to contain the pmi transgene by PCR showed positive hybridisation to a pmi DNA probe. The positive (mannose/pmi) and the negative (kanamycin) selection protocols used in this study have greatly improved transformation efficiency in almond, which were confirmed with PCR and Southern blot. This study also demonstrates that in almond the mannose/pmi selection protocol is appropriate and can result in higher transformation efficiencies over that of kanamycin/nptII selection protocols.     

Auxin pulses and a synergistic interaction between polyamines and ethylene inhibitors improve adventitious regeneration from apricot leaves and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of leaf tissues

The effect, on adventitious regeneration from apricot leaf explants and transformation of leaf tissues, of auxins pulses with NAA and 2, 4-D was tested. Addition of the polyamines putrescine and spermidine to the regeneration medium, alone or in combination with the ethylene inhibitors silver thiosulphate and aminoethoxyvinylglycine, were also tested to design a procedure that improved transformation efficiency. Spermidine at 2 mM in combination with 0.5 M aminoethoxyvinylglycine and four-day pulses with two different concentrations of 2, 4-D increased significantly shoot regeneration. Spermidine at the same concentration but in combination with 60 M silver thiosulphate and four-day pulses with 9 M 2, 4-D also increased stable transformation events and GFP-expressing calluses probably by inducing a larger amount of dividing cells where Agrobacterium transferred its T-DNA. Since regeneration from apricot leaves occurs mostly from developing calluses, it is important to obtain many GFP-expressing calluses and, given that transformation efficiencies (number of transformed shoots per total number of explants) in woody plants are generally very low, approaches that allow the optimization of T-DNA transfer and total number of transformed cells obtained, will improve probabilities of obtaining transformed shoots.     

A study of some factors affectingAgrobacterium transformation and plant regeneration ofDendranthema grandiflora Tzvelev (syn.Chrysanthemum morifolium Ramat.)

In an attempt to develop a system for producing transformed plants from explants ofDendranthema grandiflora, the susceptibility of the cultivar Super White to various wild-type strains ofAgrobacterium tumefaciens andA. rhizogenes was investigated. Tumour formation was not a reliable indicator of the ability of a related disarmed strain to mediate transformation. Following inoculation of explants with disarmedAgrobacterium strains, a number of shoots developed on selective media. However, none of these shoots were transformed. By co-cultivating stem internode explants with a mixed inoculum of wild-type and disarmed strains, it was possible to obtain a callus stably transformed withAgrobacterium carrying a disarmed T-DNA. Histological analysis of explants revealed that shoot regeneration initially occurred from the cells of the epidermis and subsequently from the cortex. However, the cells which were susceptible to T-DNA transfer were confined to the vascular tissue.     

Genetic transformation of prickly-pear cactus (Opuntia ficus-indica) by Agrobacterium tumefaciens

A system for genetic transformation of an elite prickly pear cactus (Opuntia ficus-indica L., cultivar Villa Nueva) by Agrobacterium tumefaciens was developed. Beginning with direct bacterial infection by using a hypodermic syringe to the meristematic tissue termed areoles, transgenic plants were obtained by selection with 100 mg l⁻¹ kanamycin. Transient and stable GUS activities were monitored on kanamycin-resistant shoots and regenerated plants, respectively. Genetic transformation of regenerated plants growing under selection was demonstrated by PCR and Southern blot analysis; transgene copy number in the genome of transgenic plants ranged from two to six, while the transformation frequency obtained by the system reported here was of 3.2%. This method may be useful for routine transformation and introduction of several important genes in prickly pear cactus.     

Conditions of transformation and regeneration of `Induka' and `Elista' strawberry plants

Efficiency of plants' transformation depends on many factors. The genotype, applied techniques and conditions of plant's modification and modified plant regeneration are the most important among them. In our studies regeneration and transformation conditions for two strawberry cultivars were determined and compared. Plants were transformed by Agrobacterium tumefaciens LBA₄₄₀₄ strain containing plasmid pBIN19 with nptII and gus-reporter genes. Experiment was carried out on more than 1300 leaf explants from each cultivar. Generally, `Induka' plants characterized with higher regeneration potential than `Elista'. The highest number of regenerated shoots was obtained on MS medium with 0.4 mg l ^–1 IBA and 1.8 mg l^–1 BA (3.5 and 1.8 shoots/explant for `Induka' and `Elista', respectively). After plant transformation number of regenerated, transgenic shoots was higher for `Elista' (on the average: 8.3 shoots/100 explants). The number of transgenic `Induka' shoots, obtained at the same conditions, was twice lower (4.2). Simultaneously `Induka' plants needed higher kanamycin concentration for transgenic explants selection than `Elista' (25 mg l^–1). Preliminary incubation of A. tumefaciens in LB or MS medium with acetosyringone and IAA resulted in increasing transgenic shoots number (per 100 explants: `Induka' 4.5, `Elista' 8.0–9.5 shoots). After using untreated bacteria for plants' transformation, number of transgenic plants varied (dependently on cultivar) from 3.8 to 7.0/100 explants. Applying LB or MS as basic medium as well as adding tobacco plant extract to these media did not significantly influence transformation efficiency.     

Transformation of Montmorency sour cherry (Prunus cerasus L.) and Gisela 6 (P. cerasus × P. canescens) cherry rootstock mediated by Agrobacterium tumefaciens

Sour cherry (Prunus cerasus L.) scion cv. Montmorency and rootstock cv. Gisela 6 (P. cerasus x P. canescens) were transformed using Agrobacterium tumefaciens strain EHA105:pBISN1 carrying the neomycin phosphotransferase gene (nptII) and an intron interrupted ss-glucuronidase (GUS) reporter gene (gusA). Whole leaf explants were co-cultivated with A. tumefaciens, and selection and regeneration of transformed cells and shoots of both cultivars was carried out for 12 weeks on selection medium containing 50 mg l(-1) kanamycin (Km) and 250 mg l(-1) timentin. These media were [Quoirin and Lepoivre (Acta Hortic 78:437-442, 1977)] supplemented with 0.5 mg l(-1) benzylaminopurine (BA) + 0.05 mg l(-1) indole-3-butyric acid (IBA), and woody plant medium [Lloyd and McCown (Proc Int Plant Prop Soc 30:421-427, 1980)] containing 2.0 mg l(-1) BA + 1.0 mg l(-1) IBA for cv. Montmorency and cv. Gisela 6, respectively. Seven out of 226 (3.1%) explants of cv. Montmorency and five out of 152 (3.9%) explants of cv. Gisela 6 produced 30/39 GUS- and PCR-positive shoots from the cut midribs via an intermediate callus. Southern analysis of the GUS- and PCR-positive transformants confirmed stable integration of the transgenes with 1-3 copy numbers in the genomes of seven lines of cv. Montmorency and five of cv. Gisela 6. The selected transformants have a normal phenotype in vitro.     

The use of green fluorescent protein (GFP) improves Agrobacterium-mediated transformation of ‘Spadona’ pear (Pyrus communis L.)

An efficient and reproducible system for Agrobacterium-mediated transformation of the pear (Pyrus communis L.) cultivar Spadona was developed. Leaf explants of in vitro propagated plants were cocultivated with the disarmed Agrobacterium strain EHA105 harboring the plasmid pME504, carrying the uidA-intron and nptII genes. Under selective conditions, 5% of the plantlets regenerated and were positively stained for GUS. However, most of the GUS-positive plants re-callused and subsequently died, leaving only 0.3–0.8% of these plantlets to reach maturity. In order to identify transformed shoots at early stages of regeneration, we introduced the green fluorescent protein (GFP) into the pear cultivar Spadona using the plasmid PZP carrying the nuclear-targeted GFP and nptII genes. High expression levels of GFP were detected in transgenic cells as early as 7 days after transformation. GFP marked-callii and transformed plants were observed after 14 and 24 days, respectively. Fluorescence microscopy screening of transformed plant material, under the selection of kanamycin, increased the transformation frequency to 3.0–4.0%. We conclude that the introduction of GFP improves the selection of transformed plants of Spadona pear.     

Influence of selected fruit tree pollen on life history of <Emphasis Type="Italic">Euseius stipulatus</Emphasis> (Acari: Phytoseiidae)

Euseius stipulatus (Athias-Henriot) is a predatory mite widespread in the Mediterranean region considered to be important for the biological control of spider mites in citrus orchards. Development, survival and reproduction of this phytoseiid mite feeding on seven commercially obtained pollen were studied under constant laboratory conditions (20 ± 1°C, RH 65 ± 5%, photoperiod 16L: 8D h). Mites were kept individually at rearing units with ample quantity of almond (Prunus amygdalus Batch), apple (Malus domestica Borkh), apricot (Prunus armeniaca L.), cherry (Prunus avium L.), pear (Pyrus communis L.), plum (Prunus domestica L.) and walnut (Juglans regia L.) pollen as food source. Developmental time from egg to adult varied between the several pollen tested from 8.38 ± 0.08 to 9.58 ± 0.11 days for females and from 8.23 ± 0.12 and 9.07 ± 0.12 days for males. Female longevity varied from 11.53 ± 1.22 to 51.38 ± 2.45 days, while fecundity ranged from 22.84 ± 2.30 to 43.61 ± 3.78 eggs/female. The predator was unable to reproduce when feeding on walnut pollen. Data were submitted to life table analysis and values of the intrinsic rate of increase were derived, ranging from 0.079 to 0.146 (day⁻¹). The cumulative Weibull function that was used to describe the age specific survival of females produced excellent fits to the survival data. Results show that almond, plum, cherry and apricot pollen possess higher nutritional value for E. stipulatus than pear and apple pollen and thus may contribute in sustaining and increasing the predator population in field conditions. Walnut pollen can be utilized by the predator only to survive during short periods of time when principal or alternative food sources are scarce.     

Agrobacterium tumefaciens-mediated transformation of Campanula carpatica: factors affecting transformation and regeneration of transgenic shoots

An efficient transformation system for Campanula carpatica was developed using Agrobacterium tumefaciens strains LBA4404 (harbouring the plasmid pBI121), and AGL0 (harbouring the plasmid pBEO210). This is the first report on the transformation of C. carpatica. Various factors affecting the transformation efficiency and subsequent regeneration were identified. The age of seedlings from which the explants for transformation studies were taken, and the growth conditions under which the seedlings were grown had a significant influence on the production of transformed shoots. Hypocotyls taken from 12-day-old seedlings grown in the dark were the most productive, with up to 25% of hypocotyls producing transformed shoots. Explants taken from 5-week-old seedlings produced only transformed callus. The medium used for co-cultivation and incubation also had a significant influence on transformation frequency and shoot regeneration. The cultivar Blue Uniform was more responsive than White Uniform. Both bacterial strains and plasmids were equally effective in producing transformed tissue. Transformed shoots were selected on kanamycin medium, and the presence of the uidA and nptII genes in those selected shoots was confirmed by -glucuronidase and ELISA analyses, respectively.Abbreviations BAP 6-Benzylaminopurine - NAA -Naphthalene acetic acid - TDZ Thidiazuron - BU Blue Uniform - WU White Uniform     

Transgenic peach plants (<Emphasis Type="Italic">Prunus persica</Emphasis> L.) produced by genetic transformation of embryo sections using the green fluorescent protein (GFP) as an <Emphasis Type="Italic">in vivo</Emphasis> marker

The main obstacle to genetic engineering of fruit tree species is the regeneration of transformed plantlets. Transformation events in peach (Prunus persica L.) have been reported using particle bombardment or Agrobacteriummediated transformation of immature embryos. However, the regeneration of plants from transgenic tissues is still difficult and the recovery of non-chimeric plants has not been reported to date. In this paper we describe an efficient, reliable transformation and regeneration system to produce transgenic peach plants using embryo sections of mature seeds as starting material. This represents an important advantage due to the availability of such material throughout the year. A. tumefaciens strain C58 (pMP90) containing the binary plasmid pBin19 was used as vector system for transformation. We used the Nospro-nptII-Noster cassette as a selectable marker and the CaMV35Spro-sgfp-CaMV35Ster cassette as a vital reporter gene coding for an improved version of the green fluorescent protein (sGFP). In vitro cultured embryo sections were Agrobacterium-cocultivated and, after selection, transgenic shoots were regenerated. Shoots that survived exhibited high-level of sGFP expression mainly visible in the young leaves of the apex. In vivo monitoring of GFP expression permitted an early, rapid and easy discrimination of both transgenic and escape buds. After elimination of escapes, transgenic shoots were rooted in vitro and the recovered plantlets were screened using PCR amplification. Southern analysis confirmed stable genomic integration of the sgfp transgene. The high levels of GFP expression were also maintained in the second generation of transgenic peach plants.     

Agroinfiltration of grapevine leaves for fast transient assays of gene expression and for long-term production of stable transformed cells

Agrobacterium-mediated transient assays for the analysis of gene function are used as alternatives to genetic complementation and stable plant transformation. Although such assays are routinely performed in several plant species, they have not yet been successfully applied to grapevines. We explored genetic background diversity of grapevine cultivars and performed agroinfiltration into in vitro cultured plants. By combining different genotypes and physiological conditions, we developed a protocol for efficient transient transformations of selected grapevine cultivars. Among the four cultivars analyzed, Sugraone and Aleatico exhibited high levels of transient transformation. Transient expression occurred in the majority of cells within the infiltrated tissue several days after agroinfiltration and, in a few cases, it later spread to a larger portion of the leaf. Three laboratory strains of Agrobacterium tumefaciens with different virulence levels were used for agroinfiltration assays on grapevine plants. This method promises to be a powerful tool to perform subcellular localization analyses. Grapevine leaf tissues were transformed with fluorescent markers targeted to cytoplasm (free GFP and mRFP1), endoplasmatic reticulum (GFP::HDEL), chloroplast (GAPA1::YFP) and mitochondria (β::GFP). Confocal microscope analyses demonstrated that these subcellular compartments could be easily visualized in grapevine leaf cells. In addition, from leaves of the Sugraone cultivar agroinfiltrated with endoplasmic reticulum-targeted GFP-construct, stable transformed cells were obtained that show the opportunity to convert a transiently transformed leaf tissue into a stably transformed cell line. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genetic transformation of strawberry: Stable integration of a gene to control biosynthesis of ethylene

Summary Efficient methods ofAgrobacterium-mediated transformation are described for two Pacific Northwest cultivars of strawberry (Fragaria &#x00D7;ananassa), Tristar and Totem. We report stable incorporation of a gene for control of ethylene biosynthesis, into strawberry (cultivar Totem) for the first time. Cultivar Tristar was transformed with disarmed strains ofAgrobacterium tumefaciens (A. tumefaciens), LBA4404 or EHA101, containing a binary vector with marker genesuidA andnptII. Cultivar Totem was transformed withA. tumefaciens strains EHA101 or EHA105 harboring binary vectors with selectable marker genesnptII orhpt and with a gene for S-adenosylmethionine hydrolase (SAMase) for control of ethylene biosynthesis. The frequency of transgenic shoots ranged from 12.5% to 58.8% of the original treated explants when using plasmids containing the gene encoding SAMase. Primary shoot regenerants obtained on selection medium were subjected to several iterations of tissue isolation and reculture on higher stringency selection medium for recovering uniformly transformed plantlets. Transgenic plants were confirmed by their ability to undergo rooting on medium with selection at 60 mg/liter kanamycin or 10 mg/liter hygromycin. About 95&#x2013;100% of the transformation events from different experiments were capable of profuse rooting in the presence of selection. Insertion of the SAMase gene and its integration into the strawberry genome were confirmed by Southern hybridization. About 500 plants from 250 independent transgenic events have been successfully transferred to soil for further evaluation.     

An efficient transformation system for chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.)

A reproducible and efficient transformation method was developed for Desi and Kabuli chickpeas (Cicer arietinum L.) using germinated seedlings as sources of explants. Slices derived from plumules were the most efficient at generating transformed shoots. The AGL1 Agrobacterium-treated explants were first incubated on thidiazuron-containing media, then selected using phosphinothricin. Resistant shoots were successfully transferred to soil either by grafting or in vitro rooting. In experiments each taking 4–9 months, a total of 41 confirmed transformed lines were created using embryo axis slices as source explants, giving a transformation frequency of 5.1%. Southern analysis and histochemical and leaf painting assays demonstrated integration and expression of the transgenes in the initial transformants and two generations of progeny.     

Efficient transformation and regeneration of carnation cultivars usingAgrobacterium

We have developed an efficient method for transformation and regeneration of plants from carnation,Dianthus caryophyllus L. Whole leaves fromin vitro shoot cultures were mixed withAgrobacterium, cocultivated for 5 days and then plated on 2 µg/l chlorsulfuron (CS). Regenerated shoots and shoot clusters were divided into smaller sections and plated on 3 µg/l CS for selection to produce fully transformed shoots. Geneticin (G418) and kanamycin used were not as effective selective agents as CS. All regenerated shoots were vitrified. These were normalized, rooted and transferred to the greenhouse. 100% of regenerated plants were transformed based on rooting assay, GUS assay, PCR and Southern analysis.     

Factors affecting Agrobacterium-mediated transformation and regeneration of sweet orange and citrange

Epicotyl explants of sweet orange and citrange were infected with Agrobacterium strain EHA101 harboring binary vector pGA482GG, and factors affecting the plant regeneration and transformation efficiency were evaluated. Increasing the wounded area of explants by cutting longitudinally into two halves, and optimization of inoculation density, dramatically enhanced both regeneration and transformation frequency. Inclusion of 2,4-dichlorophenoxyacetic acid (2,4-D) in the explant pretreatment medium and the co-culture medium improved the transformation efficiency by decreasing the escape frequency. More than 90% rooting frequency of transformed citrange shoots was achieved by two-step culture: first on media supplemented with auxins, and then on media without hormones. Inclusion of 20 mg l^–1 kanamycin in rooting medium efficiently discriminated transformed shoots from non-transgenic escaped shoots. Shoot grafting in vitro was used to regenerate transformed plants, due to the slow growth of most sweet orange shoots.     

An efficient protocol for genetic transformation and shoot regeneration of turmeric (Curcuma longa L.) via particle bombardment

Turmeric (Curcuma longa L.) is an important spice crop plant that is sterile and cannot be improved by conventional breeding. An efficient method for stable transformation for turmeric, C. longa L., was developed using particle bombardment. Callus cultures initiated from shoots were bombarded with gold particles coated with plasmid pAHC25 containing the bar and gusA genes each driven by the maize ubiquitin promoter. Transformants were selected on medium containing glufosinate. Transgenic lines were established on selection medium from 50% of the bombarded calluses. Transgenic shoots regenerated from these were multiplied and stably transformed plantlets were produced. Polymerase chain reaction (PCR) and histochemical GUS assay confirmed the stable transformation. Transformed plantlets were resistant to glufosinate.     

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     

Efficient Genetic Transformation of Lotus corniculatus L. and Growth of Transformed Plants in Field

An efficient protocol for shoot regeneration and genetic transformation was applied to root segments of a new Lotus corniculatus L. cultivar Bokor. The shoots, that regenerated on root segments, were inoculated with Agrobacterium rhizogenes A4M70GUS, and produced hairy roots, which on media with 0.2 mg dm⁻³ benzylaminopurine, regenerated shoots. After rooting and acclimation, the transformed plants were planted in the experimental field. Their morphological traits were compared to controls. No signs of the rol genes phenotype were present. The transformants were significantly taller than controls, while there were no significant differences in the leaf area. The glucuronidase activity and the presence of uidA gene was demonstrated in transformed plants of T₀ and in seedlings of T₁ generations. It is concluded that A. rhizogenes could be a vector of choice for the transfer of desirable genes into the bird's foot trefoil genome. This revised version was published online in July 2006 with corrections to the Cover Date.     

Different media requirements for micropropagation of apricot cultivars

Factors affecting in vitro propagation of several apricot cultivars were studied. The effect of nutrient media and BA concentration were strongly genotype dependent. Generally, best results were achieved with Quoirin and Lepoivre (1977) and a modification of WPM macronutrients (Lloyd and McCown, 1981). Optimum BA concentration was different for each cultivar but best results were obtained between 1.78 and 3.11 μM. Apricot shoots rooted well with different concentrations of IBA but most shoots showed symptoms of apical necrosis that could be overcome by dipping the shoot tips in solutions of 22.2 or 44.4 μM of BA prior to transfer to rooting medium. This revised version was published online in August 2006 with corrections to the Cover Date.