J. Brian Power

Perfluorochemicals: their applications and benefits to cell culture

The properties of perfluorochemical liquids, particularly their high gas solubility, enables them to be exploited in cell biotechnology. They can facilitate respiratory-gas delivery to prokaryotic and eukaryotic cells in culture; in some systems, they can stimulate production of biomass, yields of commercially important cellular products and, for plant systems, expression of totipotency. The recoverability, and hence recycleability, of perfluorochemicals from aqueous systems makes their routine use a commercially feasible option. This article reviews the applications and beneficial effects of perfluorochemicals in cultured microbial, animal and plant cells, including both aerobic and anaerobic systems.

Expression of a chitinase transgene in rose (Rosa hybrida L.) reduces development of blackspot disease (Diplocarpon rosae Wolf)

Blackspot, caused by the Ascomycete fungus Diplocarpon rosae, is the most widespread and pernicious disease of cultivated roses. While some species of rose possess resistance to D. rosae, none of the modern-day rose cultivars are fully resistant to the pathogen. In the current study, Biolistic gene delivery was used to introduce a rice gene, encoding a basic (Class I), chitinase into embryogenic callus of the blackspot-susceptible rose (Rosa hybrida L.) cv. Glad Tidings. The plasmid used for transformation carried the neomycin phosphotransferase (nptII) gene facilitating the selection and regeneration of transgenic plants on medium containing 250 mg/l kanamycin. Southern analysis confirmed integration of 2–6 copies of the chitinase gene into the rose genome; gene expression was confirmed by enzyme assay. Bioassays demonstrated that expression of the chitinase transgene reduced the severity of blackspot development by 13–43%. This degree of resistance to the pathogen correlated with the level of chitinase expression in the transgenic rose plants. The introduction of disease defence genes into rose provides a method of producing blackspot-resistant rose cultivars sought by breeders and growers.

SALINITY TOLERANCE AND ANTIOXIDANT STATUS IN COTTON CULTURES

This investigation focuses upon cell growth and antioxidant status in cultured cells of cotton (Gossypium herbaceum) cvs. Dhumad (salt-tolerant, TOL), H-14 (medium salt-tolerant, MED), and RAhs-2 (salt-sensitive, SEN) exposed to saline stress (50-200 mM NaCl). Mean (+/- SEM) callus fresh weight (f.wt.) and dry weight (d.wt.) gains were significantly (p <.05) greater on Murashige and Skoog (MS) [1]-based medium with 50 mM NaCl for the TOL cv. (62% and 16%, respectively) over NaCl-free controls (2020 +/- 45 and 166 +/- 4 mg, respectively); comparable differences were not observed for the MED cv. A significant (p <.05) decrease in mean f.wt. occurred with the SEN cv. exposed to 50 mM NaCl. For all cvs., there were (p <.05) reductions in mean f.wts. in medium with >or=100 mM NaCl. At 200 mM NaCl, mean f.wt. decreases were 52% (TOL), 89% (MED), and 91% (SEN), respectively. A strong correlation existed between antioxidant status and growth of cells with NaCl. Superoxide dismutase and glutathione reductase activities increased with increasing salinity in the TOL cv. to maximum values of 26.3 +/- 1.1 U mg(-1) protein and 1.05 +/- 0.01 AB(340 nm) min(-1) mg(-1) protein, respectively, at 150 mM NaCl; for the MED and SEN cvs., there were no changes in activities of these enzymes between control and salt treatments. Catalase activity decreased progressively with increasing salt concentration in all cvs. except for SEN with 100 mM NaCl, where mean catalase activity (1.75 +/- 0.04 AB(240 nm) min(-1) mg(-1) protein) was greater (p <.05) than control (1.13 +/- 0.08). Overall, cultured cotton cells provide an experimental system for investigating the role of antioxidants in salt tolerance at the cellular level.

Detection of single-copy genes in DNA from transgenic plants by nonradioactive Southern blot analysis.

Improvements to the sensitivity, speed, and reproducibility of digoxigenin (DIG)-labeled probes and chemiluminescent substrates makes these compounds increasingly popular to detect nucleic acids. High sensitivity and low background are essential in Southern blot analysis, particularly with plant DNA. This article describes a nonradioactive system to detect single-copy genes in transgenic plants. Labeling using the polymerase chain reaction (PCR) was employed to produce highly sensitive and reusable DIG-labeled probes. The background was reduced by immobilizing the DNA onto nylon filters by alkaline transfer and by minimized gel handling; the signal-to-noise ratio was improved by modification of the detection procedure.

Homoeologous pairing and recombination in backcross derivatives of tomato somatic hybrids [Lycopersicon esculentum (+) L. peruvianum]

Abstract Genomic in situ hybridization (GISH) was used to examine genome interactions in two allohexa ploid (2n=6x=72) Lycopersicon esculentum (+) L. peruvianum somatic hybrids and their seed progenies originated from subsequent backcrosses to L. esculentum. The ability of GISH to distinguish between chromatin derived from two closely related species, L. esculentum and L. peruvianum (both 2n=2x=24), allowed the precise chromosomal constitution of somatic hybrids and their backcross progenies to be unequivocally established. This enabled the interaction of species genomes to be observed at meiosis, providing clear evidence of strictly regular homoeologous pairing and the high degree of homoeologous recombination in allodiploid plants (2n=2x=24) of the BC1 generation. In segmental allodiploids of the BC2 and BC3 generations, the recombinant chromosomes continued to pair with a homoeologous partner (in the absence of a homologous one), and therefore could be stably incorporated into gametes. Chiasmata were found almost exclusively in more distal, rather subterminal, chromosome segments. A considerable proportion of meiotic recombination was detected in subterminal heterochromatic regions, often involving distal euchromatin, located in close proximity. GISH also supplied information on the extent of the overall sequence homology between the genomes of L. esculentum and L. peruvianum, indicating that despite their different breeding systems, these species may not be differentiated to a high degree genetically. The present study has demonstrated that somatic hybridization between two such closely related, but sexually incompatible or difficult to cross species, provides a way of transferring genes, via homoloeogous crossing-over and recombination, across the incompatibility barriers. Indeed, such hybrids may offer the preferred route for gene transfer, which subsequently results in more stable gene introgression than other methods.

T-DNA transfer, integration, expression and inheritance in rice: effects of plant genotype and Agrobacterium super-virulence

Summary Reproducible, efficient Agrobacterium -mediated transformation has been established for cultivars of Indica, Japonica and Javanica rice. Embryogenic calli derived from mature seed scutella were cocultivated with A. tumefaciens LBA4404 carrying (1) the binary vector pVDH65 [T-DNA encoding s-glucuronidase (gus-intron) and neomycin phosphotransferase genes; strain 0065], (2) pVDH65 and the supervirulent pTOK47 (strain 1065), or (3) the super-virulent binary vector pTOK233 [T-DNA encoding the neomycin phosphotransferase, s-glucuronidase (gus-intron) and hygromycin phosphotransferase genes]. GUS activity was observed in callus following co-cultivation with strains LBA4404(pTOK233) and 1065, but not with strain 0065. Regeneration of phenotypically normal transgenic plants occurred from 12 - 21 % , 16 - 31 %, and 10 - 19 % of transformed tissues of the cultivars Pusa Basmati 1 (Indica rice), Taipei 309 (Japonica rice), and Tinawen (Javanica rice) respectively, following co-cultivation with LBA4404 (pTOK233) and selection on hygromycin-containing medium. Single T-DNA inserts were rare in hygromycin-resistant transformants. However, T-DNA inserts were stably inherited and expressed in T1 seed generation plants of Taipei 309, with transgenes being expressed in a 3 : 1 ratio in T1 progeny, indicating the presence of active T-DNA at a single locus. Comparison of T2 generation hemizygotes and homozygotes revealed a positive correlation between transgene dosage and GUS activity.

PLANT REGENERATION BY SOMATIC EMBRYOGENESIS AND ORGANOGENESIS IN COMMERCIAL PINEAPPLE (ANANAS COMOSUS L.)

SummaryAxillary and terminal buds from suckers of Ananas comosus cv. Phuket were established on Murashige and Tucker-based (MT) medium with 2.0 mgl−1 (9.8 μM) indolebutyric acid, 2.0 mgl−1 (10.74 μM) naphthaleneacetic acid, and 2.0 mgl−1 (9.29 μM) kinetin, followed by multiplication on Murashige and Skoog-based (MS) medium containing 2.0 mgl−1 (8.87 μM) benzyladenine (BA) to provide a continuous supply of axenic shoots. Leaves, excised from such cultured shoots, produced adventitious shoots from their bases when these explants were cultured on MS medium containing 0.5 mgl−1 (2.26 μM) 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 mgl−1 (8.87 μM) BA. Embryogenic callus was produced when leaf explants were cultured on MS medium with 3.0 mgl−1 (12.42 μM) 4-amino-3,5,6-trichloropicolinic acid (picloram). Somatic embryos developed into shoots following transfer of embryogenic tissues to MS medium with 1.0 mgl−1 (4.44 μM) BA. Cell suspensions, initiated by transfer of embryogenic callus to liquid MS medium with 1.0 mgl−1 (4.14 μM) picloram or 1.0 mgl−1 (4.52 μM) 2,4-D, also regenerated shoots by somatic embryogenesis, on transfer of cells to semisolid MS medium with 1.0 mgl−1 (4.44 μM) BA. All regenerated shoots rooted on growth regulator-free MS medium, prior to ex vitro acclimation and transfer to the glasshouse. These studies provide a baseline for propagation, conservation, and genetic manipulation of elite pineapple germplasms.

Integration, expression and inheritance of two linked T-DNA marker genes in transgenic lettuce

T-DNA integration and stability were assessed in Agrobacterium-derived transgenic lettuce lines carrying a chimaeric CaMV 35S promoter-driven gus-intron gene and a chimaeric nos.nptII.nos gene. T-DNA integration was predominantly complex in transgenic plants derived from an A. tumefaciens strain carrying the supervirulent plasmid ToK47. Truncation of the right side of the T-DNA was observed in first seed generation R1 plants from one line. Complex T-DNA integration patterns did not always correlate with low transgene expression. Despite a high T-DNA copy number, ca. 30% of the lines analysed showed high transgene expression in the R1 generation. High transgene expression was stable at least to the R4 seed generation in selected high-expressing lines. Transgene expression was lost in the R2 generation in a low expressing line, while complete, heritable transgene silencing from the R0 to R2 generations was also observed in another line. A 50-fold variation in β-glucuronidase (GUS) activity and a 16-fold variation in NPTII protein content were observed between R1 plants derived from different R0 parents. Reactivation of transgene expression with 5-azacytidine in partially silenced lines indicated that low expression was associated with DNA methylation.

Plant protoplast technology: current status

Robust and reproducible protoplast-to-plant systems are crucial for underpinning genetic manipulation technology involving somatic hybridisation and transformation. Novel and effective approaches for maximising the efficiency of such protoplast cultures include supplementation of media with surfactants and artificial gas carriers, such as perfluorochemicals and haemoglobin. Physical parameters, particularly electrostimulation, also enhance the development of protoplasts and protoplast-derived cells in culture. DNA uptake into protoplasts is now a routine and universally accepted procedure in plant biotechnology for introducing and evaluating both short-term (transient) and long-term (stable) expression of genes in cells and regenerated plants. Importantly, protoplast fusion overcomes pre- and post-zygotic sexual incompatibility barriers and generates novel germplasm through new nuclear-cytoplasmic combinations. In this respect, considerable progress has been made in generating somatic hybrid plants, particularly in citrus, brassicas and potato. Isolated protoplasts are also a unique single cell system for evaluating aspects of ultrastructure, genetics and physiology, with potential for the biosynthesis of novel secondary products, including commercially-important recombinant proteins (e.g. antibodies), and as systems in toxicity screening. Recent advances in protoplast technology have benefited from advances in animal and microbial cell culture, with interesting parallels existing between these systems. Further innovations will necessitate the strengthening of interdisciplinary links in these research fields and the requirement for continued dialogue and co-operation between workers with diverse but complementary skills.

Efficient in vitro shoot regeneration responses of Phaseolus vulgaris and P. coccineus

Phaseolus vulgaris L. is the most important economic species within the genus Phaseolus, and it is grown in all parts of the world. Genetic improvement by conventional breeding has met considerable success, although production of hybrids between species within the genus has been limited due to sexual incompatibility or other evolutionary lethalities. Recent advances in tissue culture have offered the opportunity to produce cultivars which could not be obtained by conventional breeding methods, but regeneration protocols are influenced by the genotype. A standard regeneration procedure was assessed for its applicability to elite breeding lines of P. vulgaris L. and landraces of P. coccineus L. from seedling explants containing a cotyledon and a small portion of the split embryonic axis. In vitro culture response and regeneration ability varied significantly between species and amongst genotypes. P. coccineus produced more shoots per explant with a higher rooting efficiency than P. vulgaris. These significant genotype effects suggest that genetic factors are important in the response to in vitro tissue culture. Different genotypes were identified, which were more competent to in vitro culture and could produce highly responsible hybrids. This in vitro culture system carry a high potential for propagation of P. vulgaris and P. coccineus, and subsequent exploitation of hybrid forms, which could also be incorporated into somatic cell approaches to improve these species.