Strategies for signal amplification in nucleic acid detection.

Many aspects of molecular genetics necessitate the detection of nucleic acid sequences. Current approaches involving target amplification (in situ PCR, Primed in situ Labeling, Self-Sustained Sequence Replication, Strand Displacement Amplification), probe amplification (Ligase Chain Reaction, Padlock Probes, Rolling Circle Amplification) and signal amplification (Tyramide Signal Amplification, Branched DNA Amplification) are summarized in the present review, together with their advantages and limitations.

Xylem colonization of tomato by Azorhizobium caulinodans ORS571.

Tomato seedlings growing aseptically in Murashige and Skoog Medium were inoculated with Azorhizobium caulinodans ORS571 (pXLGD4), carrying the lacZ reporter gene. By microscopic analyses of inoculated tomato roots, it has been demonstrated that the xylem of tomato roots can be colonized by Azorhizobium. We discuss whether this colonization of the xylem of tomato roots by diazotrophic azorhizobia might provide a suitable niche for endophytic nitrogen fixation.

Azorhizobium caulinodans ORS571 colonizes the xylem of Arabidopsis thaliana.

Improved conditions were used for the aseptic growth of Arabidopsis thaliana to investigate whether xylem colonization of A. thaliana by Azorhizobium caulinodans ORS571 might occur. When seedlings were inoculated with ORS571 (pXLGD4) tagged with the lacZ reporter gene, nearly all of the plants showed blue regions of ORS571 colonization at lateral root cracks (LRC). The flavonoids naringenin and liquiritigenin significantly stimulated colonization of LRC by ORS571. Blue bands of ORS571 (pXLGD4) bacteria were observed histochemically in the xylem of intact roots of inoculated plants. Detailed microscopic analysis of sections of primary and lateral roots from inoculated A. thaliana confirmed xylem colonization. Xylem colonization also occurred with an ORS571 nodC mutant deficient in nodulation factors. There was no significant difference in the percentage of plants with xylem colonization or in the mean length of xylem colonized per plant between plants inoculated with either ORS571 (pXLGD4) or ORS571::nodC (pXLGD4), with or without naringenin.

Combined PI-DAPI staining (CPD) reveals NOR asymmetry and facilitates karyotyping of plant chromosomes.

This paper presents a preparative and staining procedure for plant mitotic chromosomes that uses a combination of PI (propidium iodide) and DAPI (4',6-diamidino-2-phenylindol) and which reveals a pattern of high-affinity regions for these fluorochromes. Nucleolar organiser regions (NORs), telomeres and centromeric regions exhibit high PI affinity (red), whereas other chromosomal regions exhibit high affinity for either PI (red) or DAPI (blue). NOR-bearing and other chromosomes are readily distinguished, facilitating karyotyping. The dual staining pattern was observed in all the plants tested. Aspects of NOR size, number and occurrence are discussed. A karyotype of rice metaphase chromosomes is presented, based on their fluorescent banding patterns.

Effects of glucosinolates and flavonoids on colonization of the roots of Brassica napus by Azorhizobium caulinodans ORS571.

Plants of Brassica napus were assessed quantitatively for their susceptibility to lateral root crack colonization by Azorhizobium caulinodans ORS571(pXLGD4) (a rhizobial strain carrying the lacZ reporter gene) and for the concentration of glucosinolates in their roots by high-pressure liquid chromatography (HPLC). High- and low-glucosinolate-seed (HGS and LGS) varieties exhibited a relatively low and high percentage of colonized lateral roots, respectively. HPLC showed that roots of HGS plants contained a higher concentration of glucosinolates than roots of LGS plants. One LGS variety showing fewer colonized lateral roots than other LGS varieties contained a higher concentration of glucosinolates than other LGS plants. Inoculated HGS plants treated with the flavonoid naringenin showed significantly more colonization than untreated HGS plants. This increase was not mediated by a naringenin-induced lowering of the glucosinolate content of HGS plant roots, nor did naringenin induce bacterial resistance to glucosinolates or increase the growth of bacteria. The erucic acid content of seed did not appear to influence colonization by azorhizobia. Frequently, leaf assays are used to study glucosinolates and plant defense; this study provides data on glucosinolates and bacterial colonization in roots and describes a bacterial reporter gene assay tailored easily to the study of ecologically important phytochemicals that influence bacterial colonization. These data also form a basis for future assessments of the benefits to oilseed rape plants of interaction with plant growth-promoting bacteria, especially diazotrophic bacteria potentially able to extend the benefits of nitrogen fixation to nonlegumes.

The xylem of rice (Oryza sativa) is colonized by Azorhizobium caulinodans.

Following inoculation with Azorhizobium caulinodans ORS571 (pXLGD4), lateral root development of rice and colonization of lateral root cracks by bacteria were shown to be stimulated by the flavonoid naringenin. Rice seedlings growing aseptically in the presence of naringenin were inoculated with ORS571 (pXLGD4), carrying the lacZ reporter gene. By microscopic analysis of sections of inoculated rice roots, it has been demonstrated that the xylem of rice roots can be colonized by Azorhizobium caulinodans. We discuss whether this colonization of the xylem of rice roots by azorhizobia could provide a suitable niche for endophytic nitrogen fixation.

A drop-spreading technique to produce cytoplasm-free mitotic preparations from plants with small chromosomes.

A preparation technique has been developed for plants with small chromosomes, which produces large numbers of good-quality mitotic preparations. The technique employs a hydrochloric acid treatment to hydrolyse the cytoplasm, facilitating the subsequent removal of cytoplasmic debris. The evaporative force of a methanol-based fixative is exploited to disperse the cytoplasm and to deposit the chromosomes in a single optical plane. This technique permits detailed observations of chromosome morphology and karyotyping. The mitotic preparations are also suitable for the complex analysis associated with in-situ hybridization, as in studies of genome interaction in plant hybrids.

Intergeneric somatic hybrids of rice [Oryza sativa L. (+) Porteresia coarctata (Roxb.) Tateoka].

Somatic hybrid plants were obtained following the electrofusion of rice (Oryza sativa L. cv 'Taipei 309', 2n = 2x = 24) cell suspension-derived protoplasts with non-dividing leaf protoplasts of Porteresia coarctata (2n = 4x = 48), a saline-tolerant wild species. Fusion-treated protoplasts were plated on the surface of cellulose nitrate filter membranes, overlaying Lolium multiflorum nurse cells. The nurse cells were embedded in KPR medium containing 0.5 mg l(-1) 2,4-dichlorophenoxyacetic acid and semi-solidified with SeaPlaque agarose. Putative somatic hybrid cell colonies were selected on the basis of their growth, whereby faster growing colonies were transferred preferentially to MS-based medium with 2.0 mg l(-1) kinetin, 0.5 mg l(-1)α-naphthaleneacetic acid, 30 g l(-1) sucrose and 4.0 g l(-1) SeaKem agarose to induce shoot regeneration. One hundred and nineteen regenerated plants were micropropagated clonally on MS-based medium containing 2.0 mg l(-1) 6-benzylaminopurine, 50 g l(-1) sucrose and 4.0 g l(-1) SeaKem agarose, prior to DNA extraction of plant samples. Putative somatic hybrids were initially identified by RAPD analysis, and 8 plant lines were selected for further investigation by flow cytometric ploidy determination and cytology. Plants of one line had an allohexaploid chromosome complement (2n = 6x = 72) and, following examination of its vegetative clones by GISH, were confirmed as somatic hybrids containing full chromosome complements of both O. sativa and P. coarctata.

Specific flavonoids promote intercellular root colonization of Arabidopsis thaliana by Azorhizobium caulinodans ORS571.

The ability of Azorhizobium caulinodans ORS571 and other diazotrophic bacteria to internally colonize roots of Arabidopsis thaliana has been studied. Strains tagged with lacZ or gusA reporter genes were used, and the principal colonization sites were found to be the points of emergence of lateral roots, lateral root cracks (LRCs). High frequencies of colonization were found; 63 to 100% of plants were colonized by ORS571, and 100% of plants were colonized by Herbaspirillum seropedicae. After LRCs were colonized, bacteria moved into intercellular spaces between the cortical and endodermal cell layers. Specific flavonoids, naringenin and daidzein, at 5 x 10(-5) M, significantly promoted colonization by ORS571. By using a nodC and a nodD mutant of ORS571, it was shown that neither Nod factors nor NodD are involved in colonization or flavonoid stimulation of colonization. Flavonoids did not appear to be stimulating LRC colonization by their activity as nutritional factors. LRC and intercellular colonization by H. seropedicae was stimulated by naringenin and daidzein at the same concentration that stimulated colonization by ORS571.

An improved procedure for plant regeneration from indica and japonica rice protoplasts.

Plant regeneration from protoplasts of two commercially cultivated Indian indica rice varieties, Pusa Basmati 1 and Java, has been accomplished by plating embryogenic cell suspension-derived protoplasts on the surface of filter membranes overlying agarose-embedded feeder cells of Lolium multltiflorum and Oryza ridleyi, combined with the use of a maltose-containing shoot regeneration medium. Embryogenic cell suspension cultures of Pusa Basmati 1 and Jaya were initiated from mature seed scutellum-derived calli in liquid R2 medium modified by the addition of 560 mg l(-1) of proline and 1.0 % (w/v) maltose. In both varieties, protoplast plating efficiencies up to 0.4 % were obtained, depending on the nature of the feeder cells. L. multiflorum feeder cells induced a 6-fold higher plating efficiency than feeder cells of O. ridleyi. In combination, O. ridleyi and L. multiflorum feedercells further enhanced protoplast plating efficiency. Protoplast-derived cell colonies were not obtained from protoplasts of either indica varieties in the absence of feeder cells. MS-based medium containing kinetin (2.0 mg l(-1)) and α-naphthaleneacetic acid (0.5 mg 1(-1)), together with sucrose and maltose both at 1.5 % (w/v), induced green shoot regeneration in 44 % of protoplast-derived tissues, depending on the feeder cells used for protoplast culture. In both varieties, tissues obtained using O. ridleyi feeder cells were more morphogenic than tissues obtained using L. multiflorum feeder cells, either alone or in combination with cells of O. ridleyi. In the japonica rice variety Taipei 309, this new procedure resulted in a 30-fold increase in plant regeneration from protoplasts compared to previous published procedures.

Equipment for the large-scale electromanipulation of plant protoplasts.

Electric fields are now used extensively for the genetic manipulation of plant cells through protoplast fusion and direct gene uptake. The cost of commercially available electrofusion and electroporation equipment remains prohibitive for many laboratories. This paper describes an electronic apparatus, suitable for the large-scale electrofusion and electroporation of plant protoplasts that is compatible in both function and cost with commercially available equipment.

Plant regeneration from leaf mesophyll protoplasts of the tropical woody plant, passionfruit (Passiflora edulis fv flavicarpa Degener.): the importance of the antibiotic cefotaxime in the culture medium.

Enzymatic digestion of newly expanded leaves of glasshouse-grown seedlings of passionfruit released protoplasts which exhibited highest division frequency (38.6%) when plated at a density of 1.5×10(5) ppts ml(-1) in agarose-solidified droplets of KM8P medium containing the antibiotic cefotaxime (250 µg ml(-1)). Cefotaxime was essential for sustained cell division. Protoplast-derived calli were cultured on agarsolidified MS medium with 5.0 mg H NAA, 0.25 mg l(-1) BAP and additional vitamins. These calli regenerated shoots on transfer to MS medium with 1.0 mg l(-1) BAP. Regenerated shoots were rooted in half-strength MS medium with 3.0 mg l(-1) IBA and 0.5 mg l(-1) NAA (7 d), followed by sub-culture to MS medium lacking growth regulators. The ability to regenerate plants from protoplasts of passionfruit is discussed in relation to the application of somatic cell techniques for the genetic improvement of this economically important tropical woody plant.

Quantification and comparison of chloramphenicol acetyltransferase activity in transformed plant protoplasts using high-performance liquid chromatography- and radioisotope-based assays.

Rice and petunia leaf and cell suspension protoplasts were transformed by electroporation in the presence of pDW2. This plasmid contains a chloramphenicol acetyltransferase (CAT) coding region under the control of a promoter constructed from sequences of the cauliflower mosaic virus genome. We have compared two different approaches to measuring CAT activity in this system, namely high-performance liquid chromatography (HPLC) and a radioisotope-based method. Our results show that both techniques have a similar detection limit of 10 mU CAT and (with an activity greater than 10 mU CAT) the standard error for measuring known amounts of CAT activity was less than +/- 12% for both assays. The HPLC technique, however, has a greater linear response range of 10-600 mU CAT than the radioisotope method, which has a range of 10-400 mU CAT. The HPLC assay also requires a shorter assay time. As a result of this work we believe that HPLC is a viable alternative to the radioisotope-based assay described.

Protoplast culture in high molecular oxygen atmospheres.

The effect of a molecular oxygen atmosphere on protoplast culture, compared with normal cultural procedures, was investigated for tomato, rice and jute protoplast systems. Protoplasts, in unsealed Petri dishes were cultured in a sealed 250 ml volume container in which the atmosphere was replaced with sterile molecular oxygen (O2 100% v/v) achieved by a one minute oxygen flow (10 mBar). Controls were cultured in a similar way but with a normal atmosphere (20.5% v/v oxygen). For all three species a major improvement in plating efficiency and colony formation was observed in the oxygen-enriched atmospheres. In the case of rice, cultivar Taipei 309, a significant increase in plant regeneration capacity, from protoplast-derived colonies, was observed for the high oxygen cultures.

Plant regeneration from protoplasts of wild rice (Oryza rufipogon Griff.).

Embryogenic callus initiated from basal segments of micropropagated shoots of Oryza rufipogon were used to initiate cell suspension cultures. After approximately 3 months these cultures were capable of yielding large numbers of protoplasts which underwent sustained division in agarose-solidified medium at a frequency comparable to that observed with Japonica rice protoplasts in previous studies. O. rufipogon plants were reproducibly regenerated from the protoplast-derived callus and are currently being grown to maturity. This is the first report of plant regeneration from protoplasts of a wild species of Oryza.

Root hair protoplasts ofLotus corniculatus L. (birdsfoot trefoil) express their totipotency.

Treatment of the roots of 24-48 h old seedlings of the forage legumeLotus corniculatus with 1.0% Cellulase YC, and 0.1% Pectolyase Y-23 in 4.2% mannitol solution released protoplasts from the tips of root hairs within 30-40 sec of enzyme incubation. Roots from approximately 1000 seedlings yielded 1.7×10(5) protoplasts. Ten percent of protoplasts divided to form cell colonies when cultured at 1.0×10(5) ml(-1) in droplets of KM8P medium with 0.6% Sea Plaque agarose. Colonies formed callus on UM agar medium; protoplast-derived tissues produced shoots on B5 medium containing 0.05 mg 1(-1) of BAP. Regenerated plants were phenotypically and cytologically normal (2n=2x=24±2), and produced nitrogen fixing root nodules following inoculation withRhizobium. These results confirm the totipotency of protoplasts isolated from specialised epidermal cells of seedling roots ofLotus corniculatus.

Stability of mitochondrial DNA in tissue-cultured cells of rice.

Restriction analysis of mitochondrial (mt) DNA from 3-month-old callus cultures of the cytoplasmic male sterile rice, V41A, which contains S2 or "wild abortive" cytoplasm, and its fertile maintainer, V41B, showed the same BamHI restriction profiles as mtDNA from the corresponding leaf material. Similarly, mtDNA of rice (var. Taipei 309) from leaves, a 2-month-old cell suspension (T3MS2/A), a totipotent suspension (T3MS) and a 19-month-old suspension, which had lost its protoplast regeneration ability (LB3), showed indistinguishable BamHI restriction profiles. However, clear differences in mtDNA restriction profiles were observed between LB3 and a 30-month-old suspension culture of Taipei 309 (LB1), which appeared to reflect substantial changes in the relative abundance of specific DNA sequences. Hybridisation of a maizecoxII gene probe to blots of restricted mtDNA confirmed that, while the relative abundance of certain mtDNA sequences was preserved during long-term tissue culture of rice, major changes in abundance were observed with other sequences.

Plant regeneration from protoplasts isolated from seedling cotyledons of Stylosanthes guianensis, S. macrocephala and S. scabra.

Plant regeneration from protoplasts is a prerequisite to the production of modified plants using somatic hybridization and transformation. Whole plant regeneration was achieved from protoplasts isolated from seedling cotyledons of Stylosanthes guianensis, S. macrocephala and S. scabra, three economically important species of this tropical forage legume genus. The effects of both protoplast density and protoplast culture method on cell division and plating efficiency are presented.

Production of somatic hybrid tissues following chemical and electrical fusion of protoplasts from albino cell suspensions ofMedicago sativa andM. borealis.

Protoplasts isolated from cell suspensions of albinoMedicago borealis andM. sativa were fused chemically, using two methods, and electrically. Although a small scale method of chemical fusion gave the highest fusion frequency, electrofusion was the superior technique on the basis of throughput of green somatic hybrid cell colonies. Chlorophyll-containing tissues were confirmed as being somatic hybrid by isoenzyme and cytological analyses. This is the first report of the application of albino complementation to produce somatic hybrid cells in forage legumes.