Transfer and expression of <Emphasis Type="Italic">npt</Emphasis>II and <Emphasis Type="Italic">bar</Emphasis> genes in cucumber (<Emphasis Type="Italic">Cucumis satavus</Emphasis> L.)

Summary The generation of transgenic Cucumis sativus cv. Greenlong plants resistant to phosphinothricin (PPT) was obtained using Agrobacterium tumefaciens-mediated gene transfer. The protocol relied on the regeneration of shoots from cotyledon explants. Transformed shoots were obtained on Murashige and Skoog medium supplemented with 4.4 μM 6-benzylaminopurine 3.8 μM abscisic acid, 108.5 μM adenine sulfate, and 2 mg l⁻¹ phosphinothricin. Cotyledons were inoculated with the strain EHA105 harboring the neomycin phosphotransferase II (npt II), and phosphinothricin resistance (bar) genes conferring resistance to kanamycin and PPT. Transformants were selected by using increasing concentrations of PPT (2–6 mg l⁻¹). Elongation and rooting of putative transformants were performed on PPT-containing (2 mg l⁻¹) medium with 1.4 μM gibberellic acid and 4.9 μM indolebutyric acid, respectively. Putative transformants were confirmed for transgene insertion through PCR and Southern analysis. Expression of the bar gene in transformed plants was demonstrated using a leaf painting test with the herbicide Basta. Pre-culture of explants followed by pricking, addition of 50 μM acetosyringone during infection, and selection using PPT rather than kanamycin were found to enhance transformation frequency as evidenced by transient β-glucuronidase assay. Out of 431 co-cultivated explants, 7.2% produced shoots that rooted and grew on PPT, and five different plants (1.1%) were demonstrated to be transgenic following Southern hybridization.     

Simple and Effective Regeneration of Mungbean (Vigna radiata (L) Wilczek) using Cotyledonary Node Explants

Mungbean (Vigna radiata (L) Wilczek cv ML — 267) is a recalcitrant grain legume species. Direct multiple shoots were developed from the cotyledonary node explants of 2-day-old in vitro grown seedlings of mungbean. Maximum number of shoots (an average 12.1 shoots per explant) was obtained on a medium containing MS salts, B5 vitamins and 5.0 mg l^?1 BAP. A medium with lower BAP concentration appeared suitable for rapid shoot elongation. The elongated shoots were rooted on 0.2 mg l^?1 NAA. The rooted plants were acclimatized under field conditions. The survival of the plants in the greenhouse was 90 %. Plants flowered and set seed normally.     

Agrobacterium tumefaciens-mediated transgenic plant production via direct shoot bud organogenesis from pre-plasmolyzed leaf explants of Catharanthus roseus

Production of Agrobacterium tumefaciens-mediated transgenic plants, via direct shoot bud organogenesis from leaves of Catharanthus roseus, is reported. A. tumefaciens harbouring the plasmid pBI121 with GUS gene uidA and kanamycin resistance gene nptII was used. Highest transformation efficiency of 1.4 transgenic shoots/responded explant was obtained when pre-plasmolysed leaves, pre-incubated on shoot bud induction medium for 10 days, were subjected to sonication for 30 s prior to transformation. Using a selection medium containing 50 mg kanamycin l⁻¹, transformants grew into micro-shoots and formed roots on a hormone-free half strength MS medium. The transgenic nature of the regenerated plants was confirmed by PCR amplification of uidA gene and GUS histochemical assay.     

Agrobacterium tumefaciens-mediated genetic transformation of mungbean (Vigna radiata L. Wilczek) — a recalcitrant grain legume

Agrobacterium-mediated transformation of Vigna radiata L. Wilczek has been achieved. Hypocotyl and primary leaves excised from 2-day-old in-vitro grown seedlings produced transgenic calli on B₅ basal medium supplemented with 5×10⁻⁶ M BAP, 2.5×10⁻⁶ M each of 2,4-D and NAA and 50 mg l⁻¹ kanamycin after co-cultivation with Agrobacterium tumefaciens strains, LBA4404 (pTOK233), EHA105 (pBin9GusInt) and C58C1 (pIG121Hm) all containing β-glucuronidase (gusA) and neomycin phosphotransferase II (nptII) marker genes. Transformed calli were found resistant to kanamycin up to 1000 mg^.l⁻¹. Gene expression of kanamycin resistance (nptII) and gusA in transformed calli was demonstrated by nptII assay and GUS histochemical analysis, respectively. Stable integration of T-DNA into the genome of transformed calli of mungbean was confirmed by Southern blot analysis. Transgenic calli could not regenerate shoots on B₅ or B₅ containing different cytokinins or auxins alone or in combination. However, for the first time, transformed green shoots showing strong GUS activity were regenerated directly from cotyledonary node explants cultured after co-cultivation with LBA4404 (pTOK233) on B₅ medium containing 6-benzylaminopurine (5×10⁻⁷ M) and 75 mg l⁻¹ kanamycin. The putative transformed shoots were rooted on B₅+indole-3-butyric acid (5×10⁻⁶ M) within 10–14 days and resulted plantlets subsequently developed flowers and pods with viable seeds in vitro after 20 days of root induction. The stamens, pollen grains and T₀ seeds showed GUS activity. Molecular analysis of putative transformed plants revealed the integration and expression of transgenes in T₀ plants and their seeds.     

Comparison of the effects of kanamycin and geneticin on regeneration of papaya from root tissue

Kanamycin and geneticin are commonly used for the selection of neomycin phosphotransferase II (npt II) transformed plants. Since papaya tissue is sensitive to both antibiotics, it is difficult to explore their effects on the regeneration process solely based on using non-transformed tissues. Adventitious roots derived from npt II-transgenic and non-transgenic papaya shoots in vitro were used as explants in this investigation. The effects of kanamycin and geneticin on callus formation, embryogenesis, and conversion of somatic embryos to shoots were compared. Callus growth derived from npt II-transformed root explants was apparently enhanced on kanmycin within 50–200 mg l^–1 or on geneticin within 12.5–50 mg l^–1 as compared to those on antibiotic-free controls. The percentages of npt II-transformed somatic embryo-forming callus were not significantly different (16.3–18.3%) on geneticin less than 6.25 mg l^–1 and only slightly reduced (11.2–15.7%) on geneticin within 12.5–50 mg l^–1, whereas, formation of somatic embryos was strongly suppressed on kanamycin media. Conversion rates of npt II-transformed somatic embryos to shoots were not significantly different among all kanamycin or geneticin treatments. Percentages of the callus derived from non-transformed root explants were greatly reduced on the medium containing more than 25 mg l^–1 kanamycin or geneticin, and no somatic embryos formed from untransformed callus on any kanamycin or geneticin media. Our results indicated that somatic embryogenesis of callus derived from npt II-transformed root explants of papaya was strongly inhibited by kanamycin. Thus, to regenerate npt II-transformed cells from papaya root tissue, we recommend using the lower concentration geneticin (12.5–25 mg l^–1) to avoid the adverse effects of kanamycin on embryogenesis.     

In Vitro Regeneration from Internodal Explants and Somaclonal Variation in Chickpea (Cicer arietinum L)

Protocols have been developed for the in vitro regeneration of plants from calli derived from internode explants of chickpea (Cicer arietinum L) cv Pusa-372. Callusing was induced on both B5 and MS media supplemented with different combinations and concentrations of auxins and cytokinins, but shoot regeneration was achieved only in B5 medium supplemented with 4.0 mg l^?1 IAA and 0.5 mg l^?1 BAP after serial subculture of callus on media with increasing concentration of IAA and constant concentration of BAP. Rooting could not be achieved in in vitro regenerated shoots on any one of the media tried. Complete plantlets were, therefore, developed through grafting of the in vitro regenerated shoot on established root stock. The grafting methodology was found to be highly efficient and reproducible. The somaclones developed produced viable seeds which showed variability in terms of seed colour and seed weight. Thus, the protocols developed in this study remove one important bottleneck in the development of transgenic chickpea.     

The creation of sugar beet transgenic plants expressing bar gene

The parameters of transformation using Agrobacterium tumefaciens EHA 105 for 5 domestic sorts and lines of sugar beet (Beta vulgaris L. var. saccharifera (Alef) Krass) were optimized. The system of transgenic tissue selection based on resistance to phosphinothricin, allowing to avoid the appearing of chimeric shoots among initial transformants was developed. The transgenic plants of sugar beet sorts Ramonskaya single seed 47, L’govskaya single seed 52 and RMS 73, and LBO 17 and LBO 19 lines expressing the gene of phosphinothricin acetyl transferase bar have been obtained. The resistance of these sorts and lines to the effect of phosphinothricin in vitro has been shown.     

Micropropagation of Plumbago rosea Linn.

Multiple shoot formation from the medicinal plant Plumbago rosea Linn. was induced on callus from stem segments on Murashige & Skoog media containing auxin and cytokinin. 2,4-D (2.5 mg l^-1) and kinetin (1.5 mg l^-1) added to the media gave best callus production, while BAP (2 mg l^-1) plus NAA (1.0 mg l^-1) induced shoot formation from that callus. Numerous shoots with roots could be produced by transferring shoots to media containing IBA (1.5 mg l^-1). Regenerated plantlets were transferred to pots and 60% survived.     

Efficient transgenic plant regeneration throughAgrobacterium-mediated transformation of Chickpea (Cicer arietinum L.)

Three genotypes of chickpea ICCV-1, ICCV-6 and a Desi (local) variety were tested for plant regeneration through multiple shoot production. The embryo axis was removed from mature seeds, the root meristem and the shoot apex were discarded. These explants were cultured on medium containing MS macro salts, 4X MS micro salts, I35 vitamins, 3.0 mg/1 BAP, 0.004 mg/1 NAA, 3% (w/v) sucrose and incubated at 26⁰C. The explants were transformed withAgrobacterium tumefaciens strain LBA4404 with binary vector pBI121 containing theuidA andnptIl genes. Multiple shoots were repeatedly selected with kanamycin. The selected kanamycin resistant shoots were rooted on MS medium supplemented with 0.05 mg/1 113A. The presumptive transformants histochemically stained positive for GUS. Additionally, nptll assay confirmed the expression ofnptII in kanamycin resistant plants. Transgenic plants were transferred to soil and grown in the green house.Abbreviations BAP 6-benzylamino purine - 2,4-D 2,4dichlorophenoxy acetic acid - IAA Indole acetic acid - IBA Indole butaric acid - NAA Naphthalene acetic acid     

In vitro culture and the incidence of somaclonal variation in regenerated plants of Trifolium pratense L.

Red clover (Trifolium pratense L.) cvs ‘Altaswede’ (2n = 2x = 14) and ‘Norseman’ (2n = 4x = 28) have been used to investigate tissue culture initiation, plant regeneration and the occurrence of somaclonal variation. After callus induction shoots were induced both when calli on L2 medium containing 2 mg l⁻¹ 2,4-dichlorophenoxy acetic acid (2,4-D), 2 mg l⁻¹ 6-benzylaminopurine (BA) and 2 mg l⁻¹ 6-amino-purine (AP) were subcultured on media containing naphthalene acetic acid (NAA) (0.05 mg l⁻¹) and kinetin (KIN) (0.05 or 0.5 mg l⁻¹) and when embryogenic calli were cultured and subcultured on L2 medium containing 0.002 mg l⁻¹ 4-amino-3,5,6-trichloropicolinic acid (PIC) and 0.2 mg l⁻¹ BA. Shoot tip cultures were also established to induce multiple shoots for regeneration of plants via organogenesis.Regenerants from different regeneration pathways were evaluated for chromosome number stability, morphology and several biochemical traits. Regenerated plants showed stable isozyme banding patterns for malate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucose isomerase, phosphoglucomutase and shikimate dehydrogenase, as well as their nodule leghemoglobin profiles. Variations were detected in the chromosome number of some regenerants as well as in leaflet length-to-width ratio and leaflet number. Factors related to the incidence of somaclonal variation are discussed.     

Transformation of azuki bean by Agrobacterium tumefaciens

Stable transformation and regeneration was developed for a grain legume, azuki bean (Vigna angularis Willd. Ohwi & Ohashi). Two constructs containing the neomycin phosphotransferase II gene (nptII) and either the -glucuronidase (GUS) gene or the modified green fluorescent protein [sGFP(S65T)] gene were introduced independently via Agrobacterium tumefaciens-mediated transformation. After 2 days of co-cultivation on MS medium supplemented with 100 M acetosyringone and 10 mg l^–1 6-benzyladenine, seedling epicotyl explants were placed on regeneration medium containing 100 mg l^–1 kanamycin. Adventitious shoots developing from explant calli were excised onto rooting medium containing 100 mg l^–1 kanamycin. Rooted shoots were excised and repeatedly selected on the same medium containing kanamycin. Surviving plants were transferred to soil and grown in a green house to produce viable seeds. This process took 5 to 7 months after co-cultivation. Molecular analysis confirmed the stable integration and expression of foreign genes.     

Co-production of ice nuclei and xanthan gum by transformed Xanthomonas campestris grown in sugar beet molasses

Two recombinant plasmids, expressing ice nucleation activity, were constructed and named pCPP30inaZ and pCPP38inaZ. They were transferred to the ice-negative, xanthan-producing Xanthomonas campestris pv. campestris by electroporation. The transformants were used for co-production of xanthan gum and ice nuclei from sugar beet molasses. The highest values obtained were 20 g l^–1 and 10¹⁸ ice nuclei ml^–1, respectively. The above values fulfil the criteria for industrial manipulation. This is the first report on co-formation of two products by a transformed X. campestris strain.     

Efficacy of chemical and fluorescent protein markers in studying plant colonization by endophytic non-pathogenic <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> isolates

In studying plant colonization by inoculated Fusarium oxysporum endophytes, it is important to be able to distinguish inoculated isolates from saprophytic strains. In the current study, F. oxysporum isolates were transformed with the green (GFP) and red fluorescent protein (DsRed) genes, and benomyl- and chlorate-resistant mutant isolates were also developed. The benomyl- and chlorate-resistant mutants, and the fluorescently labelled transformants, were able to grow on potato dextrose agar amended with 20 mg Benlate^® l^?1, 30 g chlorate l^?1 and 150 μg hygromycin ml^?1, respectively. Single spores of all mutants remained stable after several transfers on non-selective media. Most mutants and transformants produced colony diameters that did not differ significantly from that of their wild-type progenitors after 7 days of growth on non-selective media. Few mutants, however, had growth rates that were either slower or faster than for their wild-types. Plant colonization studies showed that root and rhizome tissue colonization by most benomyl- and chlorate-resistant mutants was similar to that of their wild-type isolates. Unlike GFP transformants, DsRed transformants were difficult to visualize in planta. Both the mutants and transformants can be used for future studies to investigate colonization, distribution and survival of biocontrol F. oxysporum endophytes in banana plants.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Perilla (<Emphasis Type="Italic">Perilla frutescens</Emphasis>)

Agrobacterium tumefaciens-mediated transformation system for perilla (Perilla frutescens Britt) was developed. Agrobacterium strain EHA105 harboring binary vector pBK I containing bar and γ-tmt cassettes or pIG121Hm containing nptII, hpt, and gusA cassettes were used for transformation. Three different types of explant, hypocotyl, cotyledon and leaf, were evaluated for transformation and hypocotyl explants resulted in the highest transformation efficiency with an average of 3.1 and 2.2%, with pBK I and pIG121Hm, respectively. The Perilla spp. displayed genotype-response for transformation. The effective concentrations of selective agents were 2 mg l⁻¹ phosphinothricin (PPT) and 150 mg l⁻¹ kanamycin, respectively, for shoot induction and 1 mg l⁻¹ PPT and 125 mg l⁻¹ kanamycin, respectively, for shoot elongation. The transformation events were confirmed by herbicide Basta spray or histochemical GUS staining of T₀ and T₁ plants. The T-DNA integration and transgene inheritance were confirmed by PCR and Southern blot analysis of random samples of T₀ and T₁ transgenic plants.     

In vitro establishment of a cycloclonal chain from nodal segments and apical buds of adult hazel (Corylus avellana L.)

Apical buds (0.5 cm) and nodal shoot segments (1.5 cm) excised from: A) field-grown branches, B) newly developed shoots from the forced outgrowth of axillary buds on A branches, C) newly developed shoots from the forced outgrowth of axillary buds on A branches submitted to cold storage were used as primary explants. Results indicate that three months cold storage greatly increases morphogenic capacity and reduces contamination and oxidation of tissues. Consequently, a multiplying chain could be easily established by culturing the tissues on a modified Murashige & Skoog (1962) medium plus 6-benzyl-aminopurine 5 mg l^-1, indole-3-acetic acid 0.01 mg l^-1 and gibberellic acid 0.1 mg l^-1. During the initiation and proliferation phases, both the proliferation and the elongation rate were significantly increased when a double-phase culture system (Viseur 1987) was used, giving rise to a higher microplant production than the one obtained using previously described methods. Plant regeneration was achieved by immersing the single microshoot's basal end in an IBA (0.1–1 mg ml^-1) solution for 10 s followed by a 20-day culture on a 1/2 MS2 medium.Abbreviations BAP 6-benzylaminopurine - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2ip 2-isopentenyladenine - MS1 and MS2 modified Murashige & Skoog media - NAA 2-maphtaleneacetic acid     

Use of the GFP reporter as a vital marker for Agrobacterium-mediated transformation of sugar beet (Beta vulgaris L.)

Molecular approaches to sugar beet improvement will benefit from an efficient transformation procedure that does not rely upon exploitation of selectable marker genes such as those which confer antibiotic or herbicide resistance upon the transgenic plants. The expression of the green fluorescent protein (GFP) signal has been investigated during a program of research that was designed to address the need to increase the speed and efficiency of selection of sugar beet transformants. It was envisaged that the GFP reporter could be used initially as a supplement to current selection regimes in order to help eliminate “escapes” and perhaps eventually as a replacement marker in order to avoid the public disquiet associated with antibiotic/herbicide-resistance genes in field-released crops. The sgfp-S65T gene has been modified to have a plant-compatible codon usage, and a serine to threonine mutation at position 65 for enhanced fluorescence under blue light. This gene, under the control of the CaMV 35S promoter, was introduced into sugar beet via Agrobacterium-mediated transformation. Early gene expression in cocultivated sugar beet cultures was signified by green fluorescence several days after cocultivation. Stably transformed calli, which showed green fluorescence at a range of densities, were obtained at frequencies of 3–11% after transferring the inoculated cultures to selection media. Cocultivated shoot explants or embryogenic calli were regularly monitored under the microscope with blue light when they were transferred to media without selective agents. Green fluorescent shoots were obtained at frequencies of 2–5%. It was concluded that the sgfp-S65T gene can be used as a vital marker for noninvasive screening of cells and shoots for transformation, and that it has potential for the development of selectable marker-free transgenic sugar beet.     

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

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

Synergistic production of L-arabinose from arabinan by the combined use of thermostable endo- and exo-arabinanases from Caldicellulosiruptor saccharolyticus

The optimum conditions for the production of l-arabinose from debranched arabinan were determined to be pH 6.5, 75 °C, 20 g l⁻¹ debranched arabinan, 42 U ml⁻¹ endo-1,5-α-l-arabinanase, and 14 U ml⁻¹ α-l-arabinofuranosidase from Caldicellulosiruptor saccharolyticus and the conditions for sugar beet arabinan were pH 6.0, 75 °C, 20 g l⁻¹ sugar beet arabinan, 3 U ml⁻¹ endo-1,5-α-l-arabinanase, and 24 U ml⁻¹ α-l-arabinofuranosidase. Under the optimum conditions, 16 g l⁻¹l-arabinose was obtained from 20 g l⁻¹ debranched arabinan or sugar beet arabinan after 120 min, with a hydrolysis yield of 80% and a productivity of 8 g l⁻¹ h⁻¹. This is the first reported trial for the production of l-arabinose from the hemicellulose arabinan by the combined use of endo- and exo-arabinanases.