High-level expression of a tobacco chitinase gene in Nicotiana sylvestris. Susceptibility of transgenic plants to Cercospora nicotianae infection

Endochitinases (E.C. 3.2.14, chitinase) are believed to be important in the biochemical defense of plants against chitin-containing fungal pathogens. We introduced a gene for class I (basic) tobacco chitinase regulated by Cauliflower Mosaic Virus 35S-RNA expression signals into Nicotiana sylvestris. The gene was expressed to give mature, enzymatically active chitinase targeted to the intracellular compartment of leaves. Most transformants accumulated extremely high levels of chitinase-up to 120-fold that of non-transformed plants in comparable tissues. Unexpectedly, some transformants exhibited chitinase levels lower than in non-transformed plants suggesting that the transgene inhibited expression of the homologous host gene. Progeny tests indicate this effect is not permanent. High levels of chitinase in transformants did not substantially increase resistance to the chitin-containing fungus Cercospora nicotiana, which causes Frog Eye disease. Therefore class I chitinase does not appear to be the limiting factor in the defense reaction to this pathogen.     

A protoplast to plant system in roses

High yields of protoplasts were isolated from embryogenic suspension cultures of Rosa persica x xanthina and Rosa wichuraiana using an enzyme mixture comprising 20 g l^-1 cellulase Onozuka R10, 1 g l^-1 Pectolyase Y-23 and 10 g l^-1 hemicellulase. Agarose-immobilized protoplasts gave the most consistent growth at a plating density of 5×10⁴ protoplasts ml^-1 on the basic medium of Kao & Michayluk (KM8p) containing 2 mg l^-1 naphthaleneacetic acid and 1 mg l^-1 benzylaminopurine. At 25°C in the dark, 0.004% of R. persica x xanthina protoplasts developed into colonies. Using similar culture conditions, but with a plating density of 9×10⁴ protoplasts ml^-1, 0.017% of R. wichuraiana protoplasts developed into colonies. On transfer of R. persica x xanthina colonies to Schenk & Hildebrandt's medium containing 3 mg l^-1 2,4-dichlorophenoxyacetic acid, globular and later stage embryos were formed. Approximately 30% of these embryos developed into plantlets on transfer to basal Schenk & Hildebrandt's medium. Further development of the plantlets took place on cellulose plugs (Sorbarods) soaked in Murashige & Skoog's medium containing 0.05 mg l^-1 naphthaleneacetic acid, 0.05 mg l^-1 indole-3-butyric acid and 0.1 mg l^-1 benzylaminopurine. Rose breeding is now open to the full range of in vitro genetic manipulation techniques involving protoplast technology.     

Requirements for plant regeneration from protoplasts of the shrubby ornamental honeysuckle,Lonicera nitida cv. Maigrun

Leaf protoplasts of axenic shoot cultures of Lonicera nitida cv Maigrun underwent sustained division to give multicellular colonies (microcalli) on a modified, ammonium-free MS (Murashige & Skoog) medium containing 0.5 mg l^-1 NAA (1-naphthaleneacetic acid), 1.0 mg l^-1 BAP (6-benzylaminopurine) and 150 mg l^-1 casein enzymatic hydrolysate. Callus was produced upon transfer of cell colonies to MS medium with 2.0 mg l^-1 NAA and 0.2 mg l^-1 BAP. About 110 days from isolation protoplast-derived shoots were regenerated on a half-strength MS medium with 0.01 mg l^-1 NAA, 5.0 mg l^-1 BAP, 0.5 mg l^-1 zeatin and a complex mixture of group B vitamins. The replacement of such mixture by 250 mg l^-1 casein enzymatic hydrolysate promoted rhizogenesis in calli, with shoot buds being subsequently regenerated from the protoplast-derived roots. Micropropagation of protoplast-derived shoots (of either origin) was difficult, due to a strong apical dominance, but could be accomplished by transferring single-node explants to half-strength MS medium with 1.5 mg l^-1 BAP. Such shoots were, in turn, successfully rooted and transferred to the glasshouse where they completed acclimatization.Abbreviations BAP 6-benzylaminopurine - CPW Power et al. (1989) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - F.P.E. final plating efficiency - f.wt. fresh weight - IAA 4-indole-3yl-acetic acid - IBA 4-indole-3yl-butyric acid - I.P.E. initial plating efficiency - MES 2-N-morpholinoethane sulfonic acid - M.P.E. intermediate plating efficiency - MS Murashige & Skoog (1962) medium - NAA 1-naphthaleneacetic acid - PVP-10 polyvinylpirrolidone - Av MW 10,000, TIBA 2,3,5-tri-iodobenzoic acid - Z zeatin     

Morphogenesis in leaf,hypocotyl and root explants of Digitalis thapsi L. cultured in vitro

The effects of the auxins 2,4-D, NAA and IAA either alone or in combination with kinetin or BA were investigated to assess the morphogenetic potential of leaf, root and hypocotyl explants of Digitalis thapsi. Calluses were obtained from the three explants in basal medium without the addition of growth regulators and in leaves, the calluses formed roots. Application of 2,4-D, NAA or BA increased callus formation. The presence of NAA induced root formation and that of BA induced shoot formation via callus interphase. Indole-3-acetic acid alone only induced the generation of roots in the hypocotyl callus. Kinetin was ineffective in all the explants tested. Combinations of NAA with kinetin or BA were more effective in inducing organogenesis in leaf explants. Optimum responses were obtained in hypocotyl and root explants by using IAA in combination with BA, the highest rate of shoot regeneration being observed in hypocotyl explants.Rooting of the differentiated shoots was readily achieved in media without growth regulators. Regenerated plantlets were transferred to soil and grew with a survival rate of 70%.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid, Kin-kinetin - NAA naphthaleneacetic acid     

Sex determination in cucumber (Cucumis sativus) as a model system for molecular biology

Floral biology and sex determination are reviewed in cucumber, one of the best studied monoecious plant systems. Sexual differentiation is controlled by genotypic and environmental factors. Sex conversion has been achieved by a variety of chemical treatments, some of which being extensively used for commercial purposes. Sex expression can be shifted in either direction: femaleness is promoted by ethylene, auxines and ethylene releasing compounds, while maleness is induced by gibberellins and chemicals counteracting ethylene action. Agrobacterium transformation affects, albeit rather nonspecifically, sex expression. An important collection of sex and floral mutants has been developed. The expression of sex genes has been shown to be under the control of modifier genes or the environment. Cloning strategies can take profit of the fact that sex conversion can be modulated alone or in combination by genetical, chemical and/or environmental parameters.     

Progress in the biotechnology of trees

An increasing world population and rise in demand for tree products, especially wood, has increased the need to produce more timber through planting more forest with improved quality stock. Superior trees are likely to arise from several sources. Firstly, forest trees can be selected from wild populations and cloned using macropropagation techniques already being investigated for fruit tree rootstocks. Alternatively, propagation might be brought aboutin vitro through micropropagation or sustained somatic embryogenesis, with encapsulation of the somatic embryos to form artificial seeds. Tree quality could be improved through increased plant breeding and it is likely that experienced gained, to date, in the breeding of fruit species will be useful in devising strategies for forest trees. Since the development of techniques to regenerate woody plants from explant tissues, cells and protoplasts, it is now feasible to test the use of tissue culture methods to bring about improvements in tree quality. Success has already been achieved for tree species in the generation of somaclonal and protoclonal variation, the formation of haploids, triploids and polyploids, somatic hybrids and cybrids and the introduction of foreign DNA through transformation. This review summarizes the advances made so far in tree biotechnology, and suggests some of the directions that it might take in the future.     

Biodiversity amongst filamentous fungi

Diversities in fungi are manifold. Fungi themselves are heterogeneous and constitute at least three unrelated major taxa. Structural diversity reflects, in most cases, adaptive and functional strategies. Diversity in nucleic acids and chemical compounds is very high in several fungal taxa. Fungi play an essential role in the function of ecosystems. The diversity of plant parasites is extremely high and species-dependent associations exist. Saprobic fungi are most important in wood and litter decay and diverse taxa comprise the main decomposers in specific successional niches. Two dominating symbiotic systems have evolved convergently in various fungal groups, notably lichens and mycorrhizas, both remarkably diverse in their heterotrophic partners.     

Gene lineage analysis in populations ofHelianthus niveus andH. petiolaris (Asteraceae)

Helianthus petiolaris andH. niveus are polytypic species which are morphologically distinct at the periphery of their ranges but intergrade in areas of sympatry.Helianthus niveus includes both annual and perennial members, whereasH. petiolaris is strictly annual. Chloroplast DNA and nuclear ribosomal DNA restriction site data were used to reconstruct the evolutionary history of populations of the two species. Cladistic analyses reveal the following: (1) neither species is monophyletic; (2) the annual habit is derived once in this complex; and (3) the region of morphological intergradation appears to be primary in origin. The significance of interbreeding versus common descent in defining species concepts is discussed in relation to the above cladistic analyses.     

Anti-predatory autecology in the geometrid larvae of Larentia clavaria pallidata

It was suggested by Heinrich that cryptic, palatable caterpillars would adopt foliar foraging techniques which would reduce the chance of their being detected. I wished to test this hypothesis further and to determine the post-discovery tactics. I found that caterpillars of Larentia clavaria pallidat, contrary to expectations, usually rested on upper surfaces of young Althaea setosa leaves but moved to the undersides when exposed to higher light intensities. This species may be effectively concealed by older canopy leaves. These larvae usually responded to predator-like stimuli by assuming a deimatic s form but rarely dropped. Dropping may be infrequent partly because younger caterpillars could not easily relocate the foot plant. I discuss, briefly the implications of host-plant selection.     

Population differences in larval hostplant use in the checkerspot butterfly, Euphydryas chalcedona

Larvae from two populations of Euphydryas chalcedona Doubleday & Hewitson (Nymphalidae) were reared on their own hostplant and that of the other population, in both pre-diapause and post-diapause instars. One population, Chico, uses Penstemon breviflorus Lindl. (Scrophulariaceae), and the other, Echo Lake, uses P. newberryi Gray. Growth rate and survival were determined for pre-diapause and post-diapause larvae from both populations on both plant species; and digestive efficiencies were calculated during the prediapause instars. The results showed that larvae from the two populations differed in their responses to the two plant species. Pre-diapause larvae from Chico performed equally well on both plant species—survival and digestive indices were not significantly different for two Penstemon species. In contrast, pre-diapause larvae from Echo Lake performed significantly worse on the non-hostplant—growth and survival were significantly lower on the non-host, P. breviflorus. In addition, comparison of digestive efficiencies for the two plants showed that larvae from Echo Lake digested P. breviflorus better than P. newberryi, but were significantly less able to convert P. breviflorus to body mass. In the post-diapause instars, larvae from Chico grew faster on the host than on the non-host. Larvae from Echo Lake grew quite slowly on both plant species and significantly more of the Echo Lake larvae returned to diapause instead of completing development.

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Résumé Des chenilles de deux populations d'E. chalcedona ont été élevées sur leur propre plante-hôte et sur celle de l'autre population, aux stades avant et après diapause. Les deux populations s'alimentent sur différentes espèces de Penstemon (Scrophulariaceae), et une population—Echo Lake—est monophage sur P. newberry, tandis que l'autre—Chico—utilise d'abord P. breviflorus, mais les chenilles après diapause sont trouvées sur au moins deux autres espèces de plantes. Les taux de croissance et de survie ont été déterminés pour des chenilles avant et après diapause pour les deux populations sur les deux plantes; les efficacités digestives ont été calculées sur les chenilles avant diapause.Les résultats ont montré que les chenilles des deux populations différaient par leur degré de spécialisation digestive sur leur plante hôte normale: les chenilles de Chico ont utilisé aussi bien les deux plantes, tandis que celles d'Echo Lake le faisaient significativement moins bien sur la plante non-hôte, par suite de l'inaptitude à la digérer. Ainsi la population oligophage est alimentairement moins spécialisée et plus capable de se débrouiller avec une plante non-hôte. Après diapause, les chenilles de Chico s'alimentaient significativement mieux sur plante hôte que non-hôte, ce qui était le cas aussi pour la population monophage. Dans l'ensemble, les chenilles de la population monophage semblaient moins capables de se débrouiller dans des conditions défavorables ou moins avantageuses.