Studies on plant regeneration from cotyledonary protoplasts in Brassica campestris

Protoplasts isolated from both 7-day-old light-grown and 4-day-old dark/dim light-grown cotyledons of four Brassica campestris varieties (Arlo, Sonja, Bunyip and Wonk Bok) were cultured in three liquid media: modified K8P, modified MS and modified Pelletier's B to compare the capacities for cell division and plant regeneration. Following cell wall regeneration the cultured protoplasts from dark/dim light-grown cotyledons of four varieties showed rapid division and high frequency of cell division compared with those isolated from light-grown cotyledons. The frequencies of cell division were significantly influenced by varieties and culture media but only in cultured protoplasts isolated from dark/dim light-grown cotyledons. The interaction between varieties and media was also significant. Cell colonies formed within 7–14 days in protoplast cultures from dark/dim light-grown cotyledons, and calli subsequently grown on a solid medium developed shoots when transferred onto a regeneration medium. Three of four tested varieties (Arlo, Sonja and Bunyip) showed shoot regeneration within 2–3 months after protoplast isolation, with a high degree of reproducibility in Arlo and Bunyip. Regenerated shoots, which were induced to root on half-strength MS medium with 0.1 mg.l^–1 IBA, survived in soil and grew to produce siliques and set viable seeds in the greenhouse. The present report is the first to document the production of regenerated plants that set seeds in Brassica campestris from cotyledonary protoplasts.Abbreviations BAP benzylaminopurine - CPW Composition of Protoplast Washing-solution - 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylenediamine-tetraacetic acid - GA₃ gibberellic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - KT kinetin - FDA fluorescein diacetate - SDS sodium dodecyl sulfate     

Gap regeneration in primary evergreen broad-leaved forests with or without a major canopy tree,Distylium racemosum,southwestern Japan: A comparative analysis

Gap regeneration was studied in a typical primary evergreen broad-leaved forest withoutDistylium racemosum, at the Kasugayama Forest Reserve, southwestern Japan and the results were compared with those from other primary evergreen broad-leaved forests in southwestern Japan, whereD. racemosum was the dominant species. Several common types of gap regeneration behavior were recognized among the major tree species and forests with or withoutD. racemosum consisted of three typical regeneration guilds which could be detected in the principal component analysis.Castanopsis cuspidata frequently regenerated in gaps from saplings recruited before gap formation in the forest withoutD. racemosum, although elsewhere, in forests withD. racemosum, it lacked advanced regeneration and regenerated in gaps from saplings recruited after gap formation. Some evergreenQuercus had their regenerations in gaps of the forest withoutD. racemosum, although elsewhere, in forests withD. racemosum, evergreenQuercus might not regenerate. The results indicate that tree species may change their regeneration behavior depending on the presence or absence of another key dominant species. This suggests that the presence and the dominance of a potential competitor induces shifts in the regeneration niche of other coexisting tree species.     

The regeneration niche of the grey mangrove (Avicennia marina): effects of salinity,light and sediment factors on establishment,growth and survival in the field

Field observations of seedlings and saplings of Avicennia marina showed patterns that correlated with salinity, light and sediment. Models that account for these observations were subsequently tested in a series of field experiments. Establishment varied within an estuary under controlled conditions but was not related to salinity or sediment type. Seedling survival was uniform over 3 years regardless of position in estuary and sediment type. Seedling densities and survival under canopies or in canopy gaps were not significantly different. However, seedling growth and density of saplings were greater in canopy gaps. Experiments involving manipulations of canopies showed no differences in seedling survival under canopies or in light gaps, but addition of slow-release fertilizer enhanced growth and survival in canopy gaps and under canopies. Long-term comparison of areas denuded of a canopy and with sediment disturbance showed enhanced establishment and survival when compared with areas with canopy gaps but with undisturbed sediments. Overall there appears to be no restriction to establishment of propagules within mangrove stands other than the supply of propagules and tidal or wave action. In contrast, recruitment to the sapling stage appears to be restricted by light and sediment resources. We suggest that propagules need to establish in a regeneration niche for seedling recruitment to the sapling stage. This differs from the view that seedlings in the under-storey are analogous to a seed pool in the soil.     

Elevated CO2 and drought alter tissue water relations of birch (Betula populifolia Marsh.) seedlings

The effect of increasing atmospheric CO₂ concentrations on tissue water relations was examined in Betula populifolia, a common pioneer tree species of the northeastern U.S. deciduous forests. Components of tissue water relations were estimated from pressure volume curves of tree seedlings grown in either ambient (350 l l^–1) or elevated CO₂ (700 l l^–1), and both mesic and xeric water regimes. Both CO₂ and water treatment had significant effects on osmotic potential at full hydration, apoplasmic fractions, and tissue elastic moduli. Under xeric conditions and ambient CO₂ concentrations, plants showed a decrease in osmotic potentials of 0.15 MPa and an increase in tissue elastic moduli at full hydration of 1.5 MPa. The decrease in elasticity may enable plants to improve the soil-plant water potential gradient given a small change in water content, while lower osmotic potentials shift the zero turgor loss point to lower water potentials. Under elevated CO₂, plants in xeric conditions had osmotic potentials 0.2 MPa lower than mesic plants and decreased elastic moduli at full hydration. The increase in tissue elasticity at elevated CO₂ enabled the xeric plants to maintain positive turgor pressures at lower water potentials and tissue water contents. Surprisingly, the elevated CO₂ plants under mesic conditions had the most inelastic tissues. We propose that this inelasticity may enable plants to generate a favorable water potential gradient from the soil to the plant despite the low stomatal conductances observed under elevated CO₂ conditions.     

Factors toxic to beech (Fagus sylvatica L.) seedlings in acid soils

The effects of highly and moderately acid soils on total biomass, biomass partitioning, fine root characteristics and nutritional status of beech seedlings (Fagus sylvatica L.) were studied in a growth chamber experiment. In Haplic Arenosols seedlings grew slowly but equally well without damage symptoms in a highly acid and a moderately acid soil horizon. The moderately acid A_h+B_w-horizon of a Eutric Cambisol was favourable to seedling growth. The fine root development was reduced in the highly acid A+B_w-horizon of a Dystric Cambisol and in the A_h+E-horizon of a Haplic Podzol, the latter of which also caused increased mortality. Seedling growth in the B₂-horizon of the Haplic Podzol was vigorous, in spite of a higher level of extractable Al and lower base saturation as compared with the A_h+E-horizon. These results are interpreted in relation to soil acidity, soil Al and nutritional status of the seedlings. We conclude that neither Al-toxicity nor nutrient deficiency cause the damage symptoms observed in the A_h+E-horizon of a Haplic Podzol and the fine root reduction in the A+B_w-horizon of a Dystric Cambisol. The damage symptoms of the PZh_A treatment seems to be more the result of H-toxicity or H-related factors other than nutrient shortage or Al-toxicity. Other pH-related toxic factors are discussed.     

Regeneration from intact and sectioned immature embryos of barley (Hordeum vulgare L.): the scutellum exhibits an apico-basal gradient of embryogenic capacity

Immature zygotic embryos from spring barley cv. Dissa were used to induce somatic embryogenenesis. Up to 158 germinated somatic embryos could be recovered per plated zygotic embryo. Critical factors for obtaining a high yield of regenerants were the size of the explant, the level of 2,4-D used for callus induction and the careful division of callus at each subculture. Use of microsections of immature embryos as explants revealed a pronounced gradient of callus formation and embryogenic response across the scutellum. Sections from the scutellar tissue at the coleoptilar end of the embryo gave the most callus and were highly embryogenic. The regeneration response of sectioned explants was comparable to that recovered from intact embryos of similar size.     

Somatic embryogenesis from callus cultures ofNardostachys jatamansi

Callus cultures ofNardostachys jatamansi DC, an endangered medicinal and aromatic plant, were established using petiole explants on MS medium supplemented with 16.1 µM -naphthaleneacetic acid and 1.16 µM kinetin. Embryogenesis in these callus cultures took place only upon sequential subculture of the callus on media having gradually decreasing auxin (16.1 to 1.34 µM NAA) and simultaneously increasing cytokinin (1.16 to 9.30 µM kinetin) concentrations over a period of 7 months. Somatic embryo to plantlet conversion took place on a medium containing 9.30 µM kinetin and 1.34 µM NAA.     

Plant regeneration through culture of isolated microspores of Triticum aestivum L.

Wheat microspores mechanically isolated from the anthers before culture and isolated from the anthers during the hole culture period in a chemically defined medium resulted in proembryos, embryos and finally plants. Of the four genotypes included, all responded with proembryos, and the two spring wheats Ciano and Walter gave rise to macroscopic embryos and plants. The frequency of embryo regeneration and the frequency of albino plants in both Ciano and Walter was in accordance with previously obtained results with anther culture derived material.Abbreviations 2,4-d 2,4-dichlorophenoxy acetic acid - NAA 1-naphthaleneacetic acid     

Plant regeneration from callus and explants of Sesbania spp.

Root, hypocotyl and cotyledon explants of Sesbania bispinosa, Sesbania cannabina, Sesbania formosa, and Sesbania sesban were cultured on Murashige and Skoog medium with benzyladenine (BA; 2.22, 4.44, 8.88 M) in combination with 2,4-dichlorophenoxyacetic acid (2,4-d; 2.26, 4.52, 9.05 M), indolebutyric acid (IBA; 0.25, 0.49, 4.92 M) or naphthaleneacetic acid (NAA; 2.69, 5.37, 10.74 M). Although all explant types developed some callus, callus occurred earliest and continued to grow fastest with hypocotyls. Media including 2.4-d or NAA gave the fastest growing callus. Callus was subcultured up to 10 times at 20-day intervals and retained a rapid growth rate. Shoots regenerated readily from both hypocotyls or cotyledons but not from roots. Shoot organogenesis was most frequent with IBA (0.25–4.92 M) in combination with BA (4.44–8.88 M) and did not occur with 2,4-d. With each species at least one medium induced shoot differentiation from more than 50 percent of the callus pieces. With one exception, media containing IBA that induced shoot organogenesis on explants also did so in callus, but media containing NAA, even when effective with explants, did not cause differentiation of callus. Shoots that differentiated were excised and cultured on MS medium without growth regulators or with IBA (2.46, 4.92, 9.84 M). Roots developed after 3–8 days on an appropriate rooting medium, often without IBA. Rooted plantlets were transplanted to pots in a greenhouse and developed into normal plants. Suitable media and protocols for initiating and subculturing callus and regenerating whole plants in vitro from callus and explants have thus been established for four species of Sesbania.     

Arrest of somatic embryo development in grapevine: histological characterization and the effect of ABA,BAP and zeatin in stimulating plantlet development

In an effort to understand the causes of arrest of somatic embryo development, generally observed in grapevine (Vitis sp.), histological studies were undertaken, using two cultivars (CH76 and 41B) which differ in their ability to develop into plants. Embryos with a high conversion rate (70%; CH76) formed a well-structured and functional shoot apex between two thread-like cotyledons. In contrast, embryos with a low conversion rate (10%; 41B) formed a normal root apex but lacked a well-structured shoot apex and developed a wide range of aberrant forms in the intercotyledonary area: uncontrolled cellular proliferation, formation of adventitious buds, over-growth of cotyledonary or leaf meristems. ABA increased the conversion rate of 41B embryos from 10% to 20%, but failed to improve embryo morphology. Zeatin and BAP promoted growth of 41B somatic embryos, but generated a high level of abnormalities and failed to improve conversion rate. Applied in combination with ABA, these PGRs increased the frequency of cotyledonary embryos, but decreased the conversion rate.