<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> and <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> transformed roots of the parasitic plant <Emphasis Type="Italic">Triphysaria versicolor</Emphasis> retain parasitic competence

Parasitic plants in the Orobanchaceae invade roots of neighboring plants to rob them of water and nutrients. Triphysaria is facultative parasite that parasitizes a broad range of plant species including maize and Arabidopsis. In this paper we describe transient and stable transformation systems for Triphysaria versicolor Fischer and C. Meyer. Agrobacterium tumefaciens and Agrobacterium rhizogenes were both able to transiently express a GUS reporter in Triphysaria seedlings following vacuum infiltration. There was a correlation between the length of time seedlings were conditioned in the dark prior to infiltration and the tissue type transformed. In optimized experiments, nearly all of the vacuum infiltrated seedlings transiently expressed GUS activity in some tissue. Calluses that developed from transformed tissues were selected using non-destructive GUS staining and after several rounds of in vivo GUS selection, we recovered uniformly staining GUS calluses from which roots were subsequently induced. The presence and expression of the transgene in Triphysaria was verified using genomic PCR, RT PCR and Southern hybridizations. Transgenic roots were also obtained by inoculating A. rhizogenes into wounded Triphysaria seedlings. Stable transformed roots were identified using GUS staining or fluorescent microscopy following transformation with vectors containing GFP, dsRED or EYFP. Transgenic roots derived from both A. tumefaciens and A. rhizogenes transformations were morphologically normal and developed haustoria that attached to and invaded lettuce roots. Transgenic roots also remained competent to form haustoria in response to purified inducing factors. These transformation systems will allow an in planta assessment of genes predicted to function in plant parasitism. Alexey Tomilov and Natalya Tomilova made an equal contribution in the paper.     

<Emphasis Type="Italic">Organogenic callus</Emphasis> as the target for plant regeneration and transformation via <Emphasis Type="Italic">Agrobacterium</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.)

A regeneration and transformation system has been developed using organogenic calluses derived from soybean axillary nodes as the starting explants. Leaf-node or cotyledonary-node explants were prepared from 7 to 8-d-old seedlings. Callus was induced on medium containing either Murashige and Skoog (MS) salts or modified Finer and Nagasawa (FNL) salts and B5 vitamins with various concentrations of benzylamino purine (BA) and thidiazuron (TDZ). The combination of BA and TDZ had a synergistic effect on callus induction. Shoot differentiation from the callus occurred once the callus was transferred to medium containing a low concentration of BA. Subsequently, shoots were elongated on medium containing indole-3-acetic acid (IAA), zeatin riboside, and gibberellic acid (GA). Plant regeneration from callus occurred 90 ∼ 120 d after the callus was cultured on shoot induction medium. Both the primary callus and the proliferated callus were used as explants for Agrobacterium-mediated transformation. The calluses were inoculated with A. tumefaciens harboring a binary vector with the bar gene as the selectable marker gene and the gusINT gene for GUS expression. Usually 60–100% of the callus showed transient GUS expression 5 d after inoculation. Infected calluses were then selected on media amended with various concentrations of glufosinate. Transgenic soybean plants have been regenerated and established in the greenhouse. GUS expression was exhibited in various tissues and plant organs, including leaf, stem, and roots. Southern and T₁ plant segregation analysis of transgenic events showed that transgenes were integrated into the soybean genome with a copy number ranging from 1–5 copies.     

Establishment of an efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated leaf disc transformation of <Emphasis Type="Italic">Thellungiella halophila</Emphasis>

Thellungiella halophila is a salt-tolerant close relative of Arabidopsis, which is adopted as a halophytic model for stress tolerance research. We established an Agrobacterium tumefaciens-mediated transformation procedure for T. halophila. Leaf explants of T. halophila were incubated with A. tumefaciens strain EHA105 containing a binary vector pCAMBIA1301 with the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene as a reporter gene. Following co-cultivation, leaf explants were cultured on selective medium containing 10 mg l⁻¹ hygromycin and 500 mg l⁻¹ cefotaxime. Hygromycin-resistant calluses were induced from the leaf explants after 3 weeks. Shoot regeneration was achieved after transferring the calluses onto fresh medium of the same composition. Finally, the shoots were rooted on half strength MS basal medium supplemented with 10 mg l⁻¹ hygromycin. Incorporation and expression of the transgenes were confirmed by PCR, Southern blot analysis and GUS histochemical assay. Using this protocol, transgenic T. halophila plants can be obtained in approximately 2 months with a high transformation frequency of 26%.     

Highly efficient <Emphasis Type="Italic">in vitro</Emphasis> adventitious shoot regeneration of peppermint (<Emphasis Type="Italic">Mentha</Emphasis> x <Emphasis Type="Italic">piperita</Emphasis> L.) using internodal explants

An in vitro regeneration system with a 100% efficiency rate was developed in peppermint [Mentha x piperita] using 5- to 7-mm-long second internode stem segments of 3-wk-old stock plants. Shoots developed at sites of excision on stem fragments either directly from the cells or via primary calluses. The optimal medium for maximum shoot initiation and regeneration contained Murashige and Skoog (MS) salts, B5 vitamins, thidiazuron (TDZ, 11.35 μM), ZT (4.54 μM), 10% coconut water (CW), 20 g l⁻¹ sucrose, 0.75% agar, adjusted to pH 5.8. A frequency of 100% shoot initiation was achieved, with an average of 39 shoots per explant. This regeneration system is highly reproducible. The regenerated plants developed normally and were phenotypically similar to Black Mitcham parents.     

<Emphasis Type="Italic">Piriformospora indica</Emphasis> and <Emphasis Type="Italic">Sebacina vermifera</Emphasis> increase growth performance at the expense of herbivore resistance in <Emphasis Type="Italic">Nicotiana attenuata</Emphasis>

A Sebacinales species was recovered from a clone library made from a pooled rhizosphere sample of Nicotiana attenuata plants from 14 native populations. Axenic cultures of the related species, Piriformospora indica and Sebacina vermifera, were used to examine their effects on plant performance. Inoculation of N. attenuata seeds with either fungus species stimulated seed germination and increased growth and stalk elongation. S. vermifera inoculated plants flowered earlier, produced more flowers and matured more seed capsules than did non-inoculated plants. Jasmonate treatment during rosette-stage growth, which slows growth and elicits herbivore resistance traits, erased differences in vegetative, but not reproductive performance resulting from S. vermifera inoculation. Total nitrogen and phosphorous contents did not differ between inoculated and control plants, suggesting that the performance benefits of fungal inoculation did not result from improvements in nutritional status. Since the expression of trypsin proteinase inhibitors (TPI), defensive proteins which confer resistance to attack from Manduca sexta larvae, incur significant growth and fitness costs for the plant, we examined the effect of S. vermifera inoculation on herbivore resistance and TPI activity. After 10 days of feeding on S. vermifera-inoculated plants, larval mass was 46% higher and TPI activity was 48% lower than that on non-inoculated plants. These results suggest that Sebacina spp. may interfere with defense signaling and allow plants to increase growth rates at the expense of herbivore resistance mediated by TPIs.     

Genetic transformation of NERICA,interspecific hybrid rice between <Emphasis Type="Italic">Oryza glaberrima</Emphasis> and <Emphasis Type="Italic">O. sativa</Emphasis>, mediated by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

We developed an efficient gene transfer method mediated by Agrobacterium tumefaciens for introgression of new rice for Africa (NERICA) cultivars, which are derivatives of interspecific hybrids between Oryza glaberrima Steud. and O. sativa L. Freshly isolated immature embryos were inoculated with A. tumefaciens LBA4404 that harbored binary vector pBIG-ubi::GUS or pIG121Hm, which each carried a hygromycin-resistance gene and a GUS gene. Growth medium supplemented with 500 mg/l cefotaxime and 20 mg/l hygromycin was suitable for elimination of bacteria and selection of transformed cells. Shoots regenerated from the selected cells on MS medium containing 20 g/l sucrose, 30 g/l sorbitol, 2 g/l casamino acids, 0.25 mg/l naphthaleneacetic acid, 2.5 mg/l kinetin, 250 mg/l cefotaxime, and 20 mg/l hygromycin. The shoots developed roots on hormone-free MS medium containing 30 mg/l hygromycin. Integration and expression of the transgenes were confirmed by PCR, Southern blot analysis, and histochemical GUS assay. Stable integration, expression, inheritance, and segregation of the transgenes were demonstrated by molecular and genetic analyses in the T₀ and T₁ generations. Most plants were normal in morphology and fertile. The transformation protocol produced stable transformants from 16 NERICA cultivars. We also obtained transformed plants by inoculation of calluses derived from mature seeds, but the frequency of transformation was lower and sterility was more frequent.     

The role of BAP in somatic embryogenesis induction from seed explants of <Emphasis Type="Italic">Arachis</Emphasis> species from Sections <Emphasis Type="Italic">Erectoides</Emphasis> and <Emphasis Type="Italic">Procumbentes</Emphasis>

Somatic embryogenesis was induced from seed explants of Arachis archeri, A. porphyrocalix (Section Erectoides) and A. appressipila (Section Procumbentes) in response to 6-benzylaminopurine (BAP). Embryo axes first developed into single shoots in response to 4.4 μM BAP. Friable embryogenic calluses were produced from the hypocotyl region of these explants in response to different BAP concentrations. Embryonic leaflets also gave rise to friable calluses, but somatic embryos were only observed in explants of A. archeri and A. appressipila. Histological analyses revealed the presence of heart-shaped, torpedo and cotyledonary stages embryos, both as isolated and fused structures. A low frequency of embryo-to-plant conversion was achieved by inducing shoot development on medium solidified with 0.5% phytagel and supplemented with 1.5% or 3% sucrose. Rooting was induced on MS supplemented with indole-3-acetic acid (IAA).     

Androgenesis in the vine cacti <Emphasis Type="Italic">Selenicereus</Emphasis> and <Emphasis Type="Italic">Hylocereus</Emphasis> (Cactaceae)

Anther culture techniques were applied to develop a methodology for producing haploid vine cactus plants. Anthers with most microspores in the middle uninucleate developmental stage from the tetraploid species Selenicereus megalanthus and the diploid species Hylocereus polyrhizus and H. undatus were cultured on basal MS medium supplemented with picloram and 6-benzyladenine (BA). H. polyrhizus and H. undatus anthers were also cultured on MS medium containing either thidiazuron (TDZ) or 2,4-dichlorophenoxyacetic acid (2,4-D). Pro-embryo development started after three days of culture. A direct androgenic embryo response was achieved in S. megalanthus with and without picloram/BA, while H. polyrhizus exhibited non-regenerative callus formation. Only a single direct androgenic embryo was obtained in H. polyrhizus (with 0.1 mg/l TDZ). H. undatus required a cold pre-treatment of 4°C for 24 h in 0.3 M D-mannitol to produce a response, but only calluses were obtained and they did not regenerate. S. megalanthus and H. polyrhizus embryos converted into plantlets after transfer to MS medium in the light. Rooted plants were acclimatized successfully, and most plants showed normal phenotypes. Flow cytometry and cytological studies revealed monoploid, haploid, dihaploid, and mixoploid plants. This study showed that androgenesis is strongly species- and culture-medium-dependent, thus revealing new perspectives in the genetics and breeding of vine cactus species. To the best of our knowledge, this is the first report on the production of monoploid, haploid and dihaploid plants in Cactaceae.     

Parasitism <Emphasis Type="Italic">versus</Emphasis> mutualism in the ant-garden parabiosis between <Emphasis Type="Italic">Camponotus femoratus</Emphasis> and <Emphasis Type="Italic">Crematogaster levior</Emphasis>

Ant-gardens represent a special type of association between ants and epiphytes. Frequently, two ant species can share the same nest in a phenomenon known as ‘parabiosis’, but the exact nature (i.e., mutualistic or parasitic) of this interaction is the subject of debate. We thus attempted to clarify the mutual costs and benefits for each partner (ants and plants) in the Crematogaster levior/Camponotus femoratus ant-garden parabiosis. The ants’ response to experimental foliar damage to the epiphytes and to the host tree as well as their behavior and interactions during prey capture were investigated to see if the purported parasitic status of Cr. levior could be demonstrated in either the ant-ant or in the ant-plant interactions. The results show that both species take part in protecting the epiphytes, refuting the role of Cr. levior as a parasite of the ant-garden mutualism. During capture of large prey Ca. femoratus took advantage from the ability of Cr. levior to discover prey; by following Cr. levior trails Ca. femoratus workers discover the prey in turn and usurp them during agonistic interactions. Nevertheless, the trade-off between the costs and benefits of this association seems then to be favorable to both species because it is known that Cr. levior benefits from Ca. femoratus building the common carton nests and furnishing protection from vertebrates. Consequently, parabiosis can then be defined as the only mutualistic association existing between ant species, at least in ant-gardens. Received 31 August 2006 ; revised 8 December 2006 ; accepted 12 December 2006     

Electrofusion of protoplasts from <Emphasis Type="Italic">Solanum tuberosum</Emphasis>, <Emphasis Type="Italic">S. bulbocastanum</Emphasis> and <Emphasis Type="Italic">S. pinnatisectum</Emphasis>

This paper discusses a number of experiments performed, involving the fusion by an electric field of mesophyll protoplasts from Solanum tuberosum cv. Bintje, S. tuberosum dihaploid clones 243, 299 and the wild tuberous disease-resistant species S. bulbocastanum and S. pinnatisectum. Three fusion experiments (S. bulbocastanum + S. tuberosum dihaploid 243, S. pinnatisectum + S. tuberosum cv. Bintje and S. pinnatisectum + S. tuberosum dihaploid 299) yielded 542 calli, the 52 ones of which produced shoots. Obtained regenerants were estimated by the flow-cytometry (FC) and RAPD analysis to determine hybrid plants.The utilisation of the FC as a useful method for detecting somatic hybrids is also discussed in this paper. The combination S. bulbocastanum + S. tuberosum dihaploid 243 led to the creation of eight somatic hybrids, the combination S. pinnatisectum + S. tuberosum cv. Bintje yielded four somatic hybrids and the combination S. pinnatisectum + S. tuberosum dihaploid 299 resulted in no hybrid regenerants. Morphology in vitro, growth vigour and production of tuber-like structures were evaluated in hybrid plants. Plants were transferred in vivo for further estimation (acclimatization, habitus evaluation and tuberization ability).     

Transformation of pollen embryo-derived explants by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> in <Emphasis Type="Italic">Hyoscyamus niger</Emphasis>

Leaf, root, stem, petiole, hypocotyl, and zygotic embryo explants, as well as pollen embryoids, and redifferentiated tissues from pollen embryoid-derived plantlets of Hyoscyamus niger L. (black henbane) were inoculated with Agrobacterium tumefaciens, harboring binary vectors (pGS Gluc1) and then cultured on media containing kanamycin. Transient -glucuronidase activity and kanamycin resistant callus formation were influenced by explant origin. Transgenic calluses were obtained at a frequency of up to 30% from all the explants tested. However, transgenic shoots were obtained only from the hypocotyl of plantlets derived from pollen embryoids. Transformation was confirmed by the ability of leaf segments to produce kanamycin resistant calluses, -glucuronidase histochemical and flurometric assays, polymerase chain reaction and Southern blot analysis. The results show that pollen embryoid-derived explants may be an alternative source for both efficient transformation and regeneration of transgenic plants in recalcitrant species.     

Evaluation of methods for celery (<Emphasis Type="Italic">Apium Graveolens</Emphasis> L.) transformation using <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> and the <Emphasis Type="Italic">bar</Emphasis> gene as selectable marker

Callus selection (CS) and the flamingo-bill explant (FB) methods were evaluated for efficacy in transformation for celery. Agrobacterium tumefaciens strains EHA105 and GV3101, each with the bar gene under the promoters NOS (pGPTV-BAR) or 35S (pDHB321.1), were used. Leaf explants were inoculated and co-cultivated for 2 d in the dark. Calluses emerged on the explants on callus medium (C), Murashige and Skoog (MS) medium + 2,4-Dichlorophenoxyacetic acid (2,4-D) (2.3 μM) + kinetin (2.8 μM) + timentin (300 mg·l⁻¹). Calluses 4- to 6-wk-old were selected for glufosinate (GS) resistance by a two step method. First, calluses were transferred to C medium + GS 0.35, 0.5, 1, 2, 5, or 10 mg·l⁻¹; calluses formed only with 0, 0.35 and 0.5 mg·l⁻¹ GS. All growing calluses from 0 and 0.35 mg·l⁻¹ and a few from 0.5 mg·l⁻¹, were divided and placed back on C + GS 0.35–0.5 mg·l⁻¹ for another 5–6 wk. Second, tolerant clones were again divided and placed on C + GS 1–50 mg·l⁻¹. When cultivar XP85 was inoculated with both strains, using pGPTVBAR, 19 glufosinate resistant (GR) callus clones were selected, but shoots regenerated only for strain EHA105 inoculations. When both of the strains (each with pDHB321.1) were inoculated on cv. XP166, 3 and 12 GR calluses occurred for EHA105 and GV3101, respectively. Using CS, a total of 34 GR callus clones were selected, and shoots were regenerated from over 50% of them on Gamborg B5 medium + 6-(γ, γ-dimethylallylamino) purine 2ip (4.9 μM) + naphthaleneacetic acid (NAA; 1.6 μM) and rooted on MS in 5–6 mo total time. Conversely, using FB with inoculation by GV3101/pDHB321.1 on cv. XP166 yielded putative transgenic celery plants confirmed by polymerase chain reaction (PCR) in just 6 wk. Transformation of the bar gene into celery was confirmed by PCR for 5 and 6 CS and FB lines, respectively. Southern blot analyses indicated 1–2 copies in CS lines and 1 copy in FB lines. Herbicide assays on whole plants with 100 and 300 mg·l⁻¹ glufosinate indicated a range of low to high tolerance for lines derived by both methods. The bar gene was found to be Mendelian inherited in one self-fertile CS derived line.     

<Emphasis Type="Italic">In vitro</Emphasis> culture of the resurrection plant <Emphasis Type="Italic">Haberlea rhodopensis</Emphasis>

The small group of resurrection plants is a unique model which could help us in further understanding of abiotic stress tolerance. The most frequently used approach for investigations on gene functions in plant systems is genetic transformation. In this respect, the establishment of in vitro systems for regeneration and micro propagation is necessary. On the other hand, in vitro cultures of such rare plants could preserve their natural populations. Here, we present our procedure for in vitro regeneration and propagation of Haberlea rhodopensis – a resurrection plant species, endemic for the Balkan region.     

Nutritional suitability and ecological relevance of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Brassica oleracea</Emphasis> as foodplants for the cabbage butterfly, <Emphasis Type="Italic">Pieris rapae</Emphasis>

The thale cress, Arabidopsis thaliana, is considered to be an important model species in studying a suite of evolutionary processes. However, the species has been criticized on the basis of its comparatively small size at maturity (and consequent limitations in the amount of available biomass for herbivores) and on the duration and timing of its life cycle in nature. In the laboratory, we studied interactions between A. thaliana and the cabbage butterfly, Pieris rapae, in order to determine if plants are able to support the complete development of the herbivore. Plants were grown in pots from seedlings in densities of one, two, or four per pot. In each treatment, one, two, or five newly hatched larvae of P. rapae were placed on fully developed rosettes of A. thaliana. In a separate experiment, the same densities of P. rapae larvae were reared from hatching on single mature cabbage (Brassica oleracea) plants. Pupal fresh mass and survival of P. rapae declined with larval density when reared on A. thaliana but not on B. oleracea. However, irrespective of larval density and plant number, some P. rapae were always able to complete development on A. thaliana plants. A comparison of the dry mass of plants in different treatments with controls (= no larvae) revealed that A. thaliana partially compensated for plant damage when larval densities of P. rapae were low. By contrast, single cress plants with 5 larvae generally suffered extensive damage, whereas damage to B. oleracea plants was negligible. Rosettes of plants that were monitored in spring, when A. thaliana naturally grows, were not attacked by any insect herbivores, but there was often extensive damage from pulmonates (slugs and snails). Heavily damaged plants flowered less successfully than lightly damaged plants. Small numbers of generalist plant-parasitic nematodes were also recovered in roots and root soil. By contrast, plants monitored in a sewn summer plot were heavily attacked by insect herbivores, primarily flea beetles (Phyllotreta spp.). These results reveal that, in natural populations of A. thaliana, there is a strong phenological mismatch between the plant and most of its potential specialist insect herbivores (and their natural enemies). However, as the plant is clearly susceptible to attack from non-insect generalist invertebrate herbivores early in the season, these may be much more suitable for studies on direct defense strategies in A. thaliana.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Zingiber petiolatum</Emphasis> (Holttum)

Summary We describe an in vitro propagation protocol for Zingiber petiolatum (Holttum), I. Theilade, a rare species from the southern part of Thailand. Fruits were surface-sterilized and seeds germinated on Murashige and Skoog medium (MS) medium supplemented with 3% sucrose. Three-month-old seedlings were used as initial plant material for in vitro propagation. Terminal buds of the plants were inoculated on MS medium containing 6-benzylaminopurine (BA; 2.2–35.5 μM) alone or in combination with 1-naphthaleneacetic acid (0.5 μM). Eight weeks after inoculation, the cultures were transferred to MS medium without plant growth regulators for 4wk. The cultures transferred from MS medium with 17.8 μM BA revealed the highest shoot induction rate of 6.1±0.7 shoots per explant. Rooting was spontaneously achieved in MS medium without plant growth regulators. Rooted plants were successfully transplanted to soil.     

Plant regeneration from leaf petioles in <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

Summary Camptothecin, produced by Camptotheca acuminata, is a pharmaceutically important compound. Transgenic technology has potential uses for the enhancement of camptothecin production; however, an efficient plant regeneration protocol for C. acuminata is not currently available. Factors that affected successful seedling germination were evaluated. The regeneration potential of various parts of seedlings was tested. Camptothecin production in regenerated plants was compared to its production in calluses and the original seedlings. Dark incubation and seed coat removal led to a higher germination rate and a higher survival rate after germination. The best shoot induction medium was found to be Gamborg's B5 medium+8.9 μM benzyladenine. Among the calluses induced from various parts of seedlings, leaf petiole calluses, leaf dise calluses, and cotyledon calluses regenerated shoots, but internode calluses did not. Furthermore, leaf petiole calluses and leaf dise calluses regenerated normal shoots, while cotyledon calluses regenerated hyperhydric shoots. Moreover, leaf petiole calluses had a higher shoot regeneration rate, 50% versus 9%, and a higher shoot number, 6.2±0.5 versus 2.0±0.3, than did leaf dise calluses on the best shoot induction medium. It took 4–6 wk to regenerate shoots after transfer into shoot induction media. Camptothecin concentration in the regenerated plants was significantly higher than that in the calluses and similar to that in the original seedlings. In conclusion, leaf petioles provide efficient plant regeneration of C. acuminata.     

<Emphasis Type="Italic">Drosophila</Emphasis><Emphasis Type="Italic">P</Emphasis> transposons of the urochordata <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Induction of streptomycin-resistant plantlets in <Emphasis Type="Italic">Solanum surattense</Emphasis> through <Emphasis Type="Italic">in vitro</Emphasis> mutagenesis

The species Solanum surattense Burm.f. has importance in ayurvedic medicine and also as vegetable. Streptomycin-resistant plantlets were induced showing chloroplast encoded mutants in S. surattense from mutagenised (ethyl methane sulphonate and gamma-rays) cotyledon explants. Chloroplast encoded – streptomycin resistant – shoots were developed from green (unbleached) sectors of the cotyledons. The streptomycin-resistant plants were similar to parental plants in morphology and ploidy level (2n=2x=24). Reciprocal crosses between streptomycin-resistant and the original streptomycin sensitive plants have shown the non-Mendelian transmission under the control of chloroplast – DNA. These antibiotic resistant plants are useful in designing biochemical selection schemes aimed at somatic hybrid/cybrid recovery in S. surattense.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

The Madagascan genus <Emphasis Type="Italic">Vaughania</Emphasis> is reduced to synonymy under <Emphasis Type="Italic">Indigofera</Emphasis> (<Emphasis Type="Italic">Leguminosae-Papilionoideae-Indigofereae</Emphasis>)

Summary  Eleven species comprising the Madagascan genus Vaughania are subsumed within the large pantropical genus Indigofera. Six new combinations are made; the remaining species were originally described in Indigofera.