Polar effects of lithium in the heart of the zebrafish Danio rerio

 The molecular signalling mechanisms that are believed to govern the patterning of the heart early in embryonic development are not well understood. We have investigated the events which occur during patterning of the vertebrate heart by exposing gastrula stage zebrafish embryos to lithium, which is known to affect the phosphoinositol signalling pathway. Treatment of embryos at 50% epiboly (5.25 h after fertilization at 28.5°C) with 0.3 m LiCl for 5–15 min, results in embryos with defects which range from mild to severe, depending on the length of time the embryos are exposed to lithium. In the heart, defects appear progressively in the inflow tract, the sinus venosus and atrium. By using an antibody that recognizes an atrium-specific isoform of myosin, our results show that lithium treatment at gastrulation specifically affects the atrium and sinus venosus, and has little obvious effect on the ventricle. Defects induced by lithium differ from those induced by retinoic acid (RA) treatment of similarly staged embryos, and suggest that lithium and RA may affect the patterning signals important for establishment of the vertebrate heart by acting on different populations of cells or by influencing different patterning pathways. Received: 8 December 1995 / Accepted: 11 April 1996     

Arbuscular mycorrhizae in a tropical sand dune ecosystem on the Gulf of Mexico

 Root samples of 37 species distributed on the beach and along a successional gradient (from mobile to stabilized areas) in a tropical sand dune system on the Gulf of Mexico showed that 97% of the species were mycorrhizal. The mycorrhizal inoculum potential of the sand from several dune areas was compared using two different bioassays. Firstly, the field rate of colonization by arbuscular mycorrhizal fungi of Chamaecrista chamaecristoides seedlings transplanted to random plots in the foredunes and in the mobile area was measured. The seedlings were harvested at intervals during 3 weeks to record mycorrhizal structures. In the mobile area, no mycorrhizal colonization was observed during the experiment. In the foredunes, hyphae and external mycelium were present in 40% of the seedlings as early as 8 days after transplanting. After 15 days, arbuscules and vesicles were observed in 60 and 20% of the seedlings, respectively, and after 21 days, 100, 46 and 20% of the seedlings showed hyphae, arbuscules and vesicles, respectively. Secondly, maize seedlings were transplanted to pots previously filled with sand from the foredunes, mobile dunes, grassland and a Dyphisa robinoides shrub area. After 1 month, the lowest mycorrhizal inoculum potential was recorded for the mobile dunes and the highest for the shrub area. As expected, mycorrhizal inoculum potential increased with dune stabilization. Accepted: 17 July 1996     

Effects of seasonal rainfall on radial growth in two tropical tree species

 Seasonal drought may limit primary productivity in most of the tropics, but the determinants of tree growth are not well known. A 10-year study of the deciduous trees Cochlospermum vitifolium (Willd.) Spreng. (Cochlospermaceae) and Cnidoscolus spinosus Lundell (Euphorbiaceae) in southwestern México showed radial growth to be highly correlated (both r>0.85) only with precipitation during an interval of <2 months in the mid-wet season. Growth was not affected by total annual precipitation or by an early starting or late ending of the wet season, or by heavy rainfall in the dry season. Annual mean girth increments ranged from 0.03 to 3.31 cm and −0.1 to 2.01 cm, respectively. The best model for growth (r ²>0.85) was a linear combination of mid-summer precipitation (positive coefficient) and total precipitation over the previous 2 years (negative coefficient). Comparison with other species showed heterogeneous responses of wood production to climate variation, and suggests that the range of functional types of dry forest trees is still unknown. Received: 9 September 1996 / Accepted: 4 March 1997     

Dew and its effect on two heliophile understorey species of a tropical dry deciduous forest in Mexico

 A series of horizontal and vertical measurements of dew deposition and dew duration were carried out in a tropical dry deciduous forest in western Mexico (19° 30′ N, 105° 03′ W). The effect of dew on transpiration in heliophile species was also investigated. The amount of dew was very variable with no temporal or spatial pattern. The amount of dew measured at two horizontal transects (maximum and minimum) (at heights of 0.20 m and 1.30 m above ground level) was from 0.014 to 0.203 mm and from 0.013 to 0.061 mm in the middle and at the end of the dry season, respectively. Dew deposition at different vertical levels (0.50–12.5 m height) ranged from 0.04 to 0.36 mm. The duration of dew formation ranged between 60 and 129 min after sunrise above the canopy (a height of 11 m), and between 259 and 290 min after sunrise at a height of 2 m. Daily transpiration rates were 883 and 632 g m^–2 d^–1 in Coccoloba liebmannii and 538 and 864 g m^–2 d^–1 in Jacquinia pungens in January and April, respectively. Transpiration was restricted from sunrise to early afternoon in April, as a result of the pronounced midday closure of stomata in both species. The reduction of transpiration by dew ranged from 13.2 to 50.1 g m^–2 d^–1 and from 4.5 to 77.7 g m^–2 d^–1 for C. liebmannii and J. pungens, respectively. Dew can play an important role in enhancing the survival of heliophile species in the dry season by reducing transpiration rates during the morning. Received: 30 April 1998 / Accepted: 14 December 1998     

Mapping of the Rf-3 nuclear fertility-restoring gene for WA cytoplasmic male sterility in rice using RAPD and RFLP markers

The cytoplasmic male sterility (CMS) of wild-abortive (WA) cytoplasm has been widely used for breeding hybrid rice. Two restorer genes for the CMS have been found by traditional genetic analysis. To tag the restorer genes we used a set of near-isogenic lines (NILs) of Zhenshan 97 carrying different genotypes for fertility restoration from IR24, to perform RAPD analysis. From the survey of 720 random primers, six RAPD markers were identified to be associated with Rf-3. Three of these OPK05-800, OPU10-1100 and OPW01-350, were mapped on chromosome 1. Two populations from the crosses between Zhenshan 97 A and a near-isogenic restorer line ZSR21 and between Zhenshan 97 A and IR24 were used for mapping Rf-3. The three RAPD markers and three RFLP markers, RG532, RG140 and RG458, were found to be closely linked to Rf-3 in the two populations. The same location of Rf-3 was also found in a population from the cross of IR58025 A//IR36/IR58025 B. At the RG532 locus, different alleles were found between two CMS lines, Zhenshan 97 A and IR58025 A, and between two restorer lines, IR24 and IR36. The use of these molecular markers closely linked to Rf-3 in facilitating the development of hybrid rice is discussed. Received: 3 January 1996 / Accepted: 17 May 1996     

Experimental evaluation of the potential of tropical germplasm for temperate maize improvement

 Commercial maize (Zea mays L.) in the USA has a restricted genetic base as newer hybrids are largely produced from crosses among elite inbred lines representing a small sample (predominantly about 6- to 8-base inbreds) of the Stiff stalk and Lancaster genetic backgrounds. Thus, expansion of genetic diversity in maize has been a continuous challenge to breeders. Tropical germplasm has been viewed as a useable source of diversity, although the integration of tropical germplasm into existing inbred line and hybrid development is laborious. The present study is an evaluation of the potential of tropical germplasm for temperate maize improvement. All possible single-, three-way-, and double-cross hybrids among three largely temperate and three temperate-adapted, all-tropical inbred lines were evaluated in yield-trial tests. Single-cross hybrids containing as much as 50–60% tropical germplasm produced 8.0 t ha^-1 of grain yield, equivalent to the mean yield of the commercial check hybrids. On the other hand, three-way and double-cross hybrids with the highest mean yield contained lower amounts of tropical germplasm, 10–19% and 34–44%, respectively. Overall, hybrids containing 10–60% tropical germplasm yielded within the range of the commercial hybrid checks. Hybrids with more than 60% tropical germplasm had significantly lower yields, and 100% tropical hybrids yielded the least among all hybrids evaluated. The results indicate that inbred lines containing tropical germplasm are not only a useful source to expand the genetic diversity of commercial maize hybrids, but they, also are competitive in crosses with temperate materials, producing high-yielding hybrids. These experimental hybrids exhibited good standability (comparable to the commercial check hybrids) but contained 1–2% higher grain moisture, leading to delayed maturity. Recurrent selection procedures are being conducted on derivatives of these materials to extract lines with superior yield, good standability, and reduced grain moisture which can be used for commercial exploitation. Received: 26 January 1998 / Accepted: 14 July 1998     

Production of transgenic tropical maize with cryIAb and cryIAc genes via microprojectile bombardment of immature embryos

To enhance the level of resistance to insects in tropical maize germplasm we have developed techniques to successfully transform elite tropical maize inbred based on the activity of specific cryI proteins against four major maize pests – corn earworm, fall armyworm, southwestern corn borer and sugarcane borer. Constructs containing cryIAb or cryIAc synthetic genes were used. To generate transgenic plants we have established methods for biolistic bombardment and the selection and regeneration of immature embryos and calli from the elite tropical lines CML72, CML216, CML323, CML327 and hybrids. Transgenic plants resistant to the herbicide Basta^TM contained the bands for the cry, bar and gus genes as detected by Southern blot analyses. A simple leaf bioassay presented varying levels of resistance to Southwestern corn borer of transgenic tropical maize carrying the cryIAc gene. Analyses of the progenies confirmed the sexual transmission of the introduced genes and their stable expression. Received: 25 September 1998 / Accepted: 27 October 1998     

Nitrogenase activity and N2 fixation are stimulated by elevated CO2 in a tropical N2-fixing tree

 Seeds of Gliricidia sepium, a fast-growing woody legume native to seasonal tropical forests of Central America, were inoculated with N₂-fixing Rhizobium bacteria and grown in environmentally controlled glasshouses for 67–71 days under ambient CO₂ (35 Pa) and elevated CO₂ (70 Pa) conditions. Seedlings were watered with an N-free, but otherwise complete, nutrient solution such that bacterial N₂ fixation was the only source of N available to the plant. The primary objective of our study was to quantify the effect of CO₂ enrichment on the kinetics of photosynthate transport to nodules and determine its subsequent effect on N₂ fixation. Photosynthetic rates and carbon storage in leaves were higher in elevated CO₂ plants indicating that more carbon was available for transport to nodules. A ¹⁴CO₂ pulse-chase experiment demonstrated that photosynthetically fixed carbon was supplied by leaves to nodules at a faster rate when plants were grown in elevated CO₂. Greater rates of carbon supply to nodules did not affect nodule mass per plant, but did increase specific nitrogenase activity (SNA) and total nitrogenase activity (TNA) resulting in greater N₂ fixation. In fact, a 23% increase in the rate of carbon supplied to nodules coincided with a 23% increase in SNA for plants grown in elevated CO₂, suggesting a direct correlation between carbon supply and nitrogenase activity. The improvement in plant N status produced much larger plants when grown in elevated CO₂. These results suggest that Gliricidia, and possibly other N₂-fixing trees, may show an early and positive growth response to elevated CO₂, even in severely N-deficient soils, due to increased nitrogenase activity. Received: 27 February 1996 / Accepted: 19 June 1996     

Flow cytometric analysis of the chromosomes and stability of a wheat cell-culture line

A rapidly growing, long-term suspension culture derived from Triticum aestivum L. (wheat) was synchronized using hydroxyurea and colchicine, and a chromosome suspension with chromosomes was made. After staining with the DNA-specific fluorochromes Hoechst 33258 and Chromomycin univariate and bivariate flow-cytometry histograms showed 15 clearly resolved peaks corresponding to individual chromosome types or groups of chromosomes with similar DNA contents. The flow karyotype was closely similar to a histogram of DNA content measurements of Feulgen-stained chromosomes made by microdensitometry. We were able to show the stability of the flow karyotype of the cell line over a year, while a parallel subculture had a slightly different, stable, karyotype following different growth conditions. The data indicate that flow cytometric analysis of plant karyotypes enables accurate, statistically precise chromosome classification and karyotyping of cereals. There was little overlap between individual flow-histogram peaks, so the method is useful for flow sorting and the construction of chromosome specific-recombinant DNA libraries. Using bivariate analysis, the AT:GC ratio of all the chromosomes was remarkably similar, in striking contrast to mammalian flow karyotypes. We speculate about a fundamental difference in organization and homogenization of DNA sequences between chromosomes within mammalian and plant genomes. Received: 24 April 1996 / Accepted: 24 May 1996     

Target-cell-induced anergy in natural killer cells: suppression of cytotoxic function

 Our earlier studies have demonstrated that natural killer (NK) cells are the effectors that participate during the spontaneous regression of AK-5 tumour in syngeneic hosts. We have shown that the tumour cells are killed by necrosis and apoptosis. In this study, we have examined the induction of functional anergy in NK cells following coculture with fixed AK-5 tumour cells at high ratio. NK cells, upon coculture with fixed AK-5 cells (1:1 ratio), showed loss of cytotoxic function against both AK-5 (antibody-dependent cell cytotoxicity) as well as YAC-1 targets. The response of these cells to the activation by recombinant interleukin-2 and recombinant interferon γ was poor. Induction of tumour necrosis factor α (TNFα) secretion was observed after coculture of NK cells with fixed AK-5 cells. The cocultured cell supernatant inhibited the cytotoxic activity of NK cells, which was partially restored with anti-TNFα antibody. In addition, NK cells, after treatment with fixed tumour cells showed overexpression of the Fas receptor. We have also observed induction of apoptosis in cocultured NK cells. These studies suggest that the fixed tumour cells (antigen) at high ratio are able to suppress NK cell function as well as induce death in NK cells. Received: 16 September 1999 / Accepted: 13 January 2000     

Effects of irradiance and spectral quality on seedling development of two Southeast Asian Hopea species

 Seedling developmental responses to understory shade combine the effects of reductions in irradiance and changes in spectral quality. We studied the seedling development of two Southeast Asian dipterocarp trees in response to differences in irradiance (photosynthetic photon flux density, PPFD) and spectral quality (red to far-red ratio, R:FR). The two species, Hopea helferei and H. odorata, are taxonomically closely related but differ in their ecological requirements; H. helferei is more drought-tolerant and typically grows in more open habitats. Seedlings were grown in six different replicated shadehouse treatments varying in percentage of solar PPFD and R:FR. The two species differed in the influence of light variables on most seedling characters, particularly for final height, internode distance, branch/trunk internodes, stem length/mass, leaf area/stem length, petiole length, and growth/mol of photons received. Most of the characters in both taxa were primarily influenced by PPFD, but spectral quality also influenced some characters – more so for H. odorata. The latter species grew more rapidly, particularly in the low PPFD treatments, and its leaves were capable of higher photosynthesis rates. However, growth in H. helferei was not reduced in direct sunlight. The growth of this taxon may be constrained by adaptations, particularly in leaves, for drought tolerance. Received: 14 April 1996 / Accepted: 8 October 1996     

Cell-specific density of symbiotic dinoflagellates in tropical anthozoans

 Symbiotic dinoflagellates are abundant in the endoderm cells of tropical marine anthozoans, but the cell-specific density (CSD) of symbionts has not yet been investigated. In this study we used mechanical and enzymatic methods of maceration, and staining with substrate-specific fluorochromes, to observe a large number of individual host cells from 33 species of tropical anthozoans collected in Florida, Hawaii and Jamaica or cultured in Monaco. In the majority of species, most of the host cells contained a single algal cell (singlet). Host cells with two or more (up to six) algae were much less abundant. The average CSD for the 33 species was 1.54±0.30 (range 1.11 to 2.19). Singlets arranged in a monolayer can account for the areal density of algae observed in many anthozoans. The dinoflagellates occupy most of the interior of macerated host cells, leaving the host cytoplasm and cell membrane as a thin outer layer, often unresolvable by light microscopy. This spatial arrangement may favor diffusion and transport of CO₂, bicarbonate ions, and nutrients from the environment to the algae. The effect of nutrient enrichment on CSD was determined by exposing eleven species to chronically elevated levels of ammonium-N. After four weeks all species exhibited a dramatic increase in algal mitotic index and CSD. The potential consequences of environmentally induced increases in CSD in tropical anthozoans are discussed in terms of the decreased cell-specific photosynthesis (CO₂ limitation) and decreased rates of calcification observed in other studies. Accepted: 16 February 1998     

Levels of genetic diversity at different stages of the domestication cycle of interior spruce in British Columbia

Concerns over the reductionist nature of the domestication of forest-tree species focus on the possibility of potential genetic erosion during this process. To address these concerns, genetic diversity assessments in a breeding zone the Province of British Columbia “interior” spruce (Picea glauca×engelmanni) program was conducted using allozyme markers. Genetic-variation comparisons were made between natural and production (seed orchard) populations as well as seed and seedling crops produced from the same breeding zone’s seed orchard. The natural population sample consisted of a total of 360 trees representing three stands within each of three watersheds present in the Shuswap-Adams low-elevation zone of interior British Columbia. Small amounts of genetic differentiation were observed among the nine natural populations (4%) and this was attributable to extensive gene flow Consequently, the sum of these nine populations was considered as a baseline for the genetic variation present in the breeding zone. The comparisons between the seed orchard and the breeding zone produced a similar percentage of polymorphic loci while the expected hetrozygosity (0.207 vs 0.210) and the average number of alleles per locus (2.7 vs 2.4) were slightly lower in the seed orchard. A total of seven natural populations’ rare alleles were not present in the orchard population, while one allele was unique to the orchard. The %P increased to 70.6% in the seedlot, but dropped to the natural populations level (64.7%) in the plantation. The observed increase in %P was a result of pollen contamination in the orchard. It is suspected that the reduction in the plantation was caused by an unintentional selection in the nursery. Simulated roguing in the orchard did not drastically reduce even if up to 50% of the orchard’s clones were rogued. However, roguing was associated with a reduction in the average number of alleles per locus (i.e., sampling effect). Received: 2 January 1996 / Accepted: 24 May 1996     

Presence of arbuscular mycorrhizas in typically ectomycorrhizal host species from Cameroon and New Zealand

 Ectomycorrhizas (EcM) and arbuscular mycorrhizas (AM) were screened for in saplings of 14 EcM tree species from the N'Dupé and Korup National Park rainforests, SW Cameroon, belonging to Caesalpiniaceae and Uapacaceae. The pattern of EcM and AM colonisation of a dual mycorrhizal species from this rainforest (Uapaca staudtii, Uapacaceae) was compared with dual EcM/AM colonisation of Leptospermum scoparium (Myrtaceae) from New Zealand. Both species were collected in a range of habitats. EcM and AM colonisation differed among species in the Korup National Park rainforest: 12 species belonging to the Caesalpiniaceae (Amherstieae) were consistently EcM, and AM structures occurred occasionally in six of them; two other species belonging to Caesalpiniaceae (Afzelia bipindensis) and Uapacaceae (U. staudtii) were dual mycorrhizal with variable levels of colonisation by both EcM and AM fungi. EcM and AM dual colonisation varied with both habitat and identity of the partners. The presence of EcM fungi in most of the root samples of U. staudtii and a negative relationship between AM and EcM colonisation within the same root system suggested a greater EcM affinity of this species. In contrast, most root samples of L. scoparium were colonised by AM, but only a few by EcM. Genuine dual EcM/AM associations in root samples of U. staudtii where the two mycorrhizal types co-occurred could be attributed to an AM-EcM succession. However, differences between predicted and observed frequencies of genuine dual EcM/AM associations in several samples of both U. staudtii and L. scoparium indicated that other factors influenced dual EcM/AM associations. The results of this study showed the importance of the identity of the host species in determining the pattern of dual EcM and AM colonisation. Accepted: 18 September 1998     

Arbuscular mycorrhizal infection in Cyperaceae and Gramineae from natural,disturbed and restored savannas in La Gran Sabana,Venezuela

 The mycorrhizae of a tropical savanna growing in oligotrophic and stony soils were compared with those of a disturbed area that had been reclaimed with introduced species and of an area that was disturbed but not revegetated. All were compared with natural regeneration in a savanna that had been disturbed 12 years previously. Arbuscular mycorrhizae (AM) were common in savannas. Cyperaceae species, which were codominant with Graminaea, showed high levels of infection frequency (45%) like the Gramineae (61%). Arbuscules observed in the Cyperaceae indicated functionality. There were few plants in disturbed, nonrevegetated sites, but those present had AM. Observations of roots from soil monoliths showed that AM were present in disturbed areas, but compared with natural, succesional and revegetated savanna had a lower infection frequency (48–59% vs 75%), lower intensity (10–15% vs 25%) and a lower percentage of arbuscules (0.7–0.8% vs 2.3%). The percentage of vesicles was also lower in succesional savanna than in natural savanna (1.6% vs 4.8%). The revegetated site had the highest percentage of vesicles (6.6%). Although a high frequency of mycorrhizal infection has been reestablished in disturbed areas, the intensity and structure of the infection suggests that mycorrhizal function has not been restored to the original levels. We hypothesize that neither plants nor fungi have adapted to the new edaphic conditions. Accepted: 10 July 1995     

A comparison of the development and metabolic activity of mycorrhizas formed by arbuscular mycorrhizal fungi from different genera on two tropical forage legumes

 Two glasshouse experiments were done to assess the development and metabolic activity of mycorrhizas formed by isolates of arbuscular mycorrhizal fungi (AMF) from three different genera, Acaulospora, Gigaspora and Glomus on either Pueraria phaseoloides L. or Desmodium ovalifolium L. plants. The second of the two experiments included three levels of a localised phosphate source in the pots. Alkaline phosphatase (ALP), stained histochemically in the intra-radical mycelium (IRM) of AMF over sequential harvests, did not provide a direct marker for the efficiency of AMF in mobilising phosphorus (P) for plant growth and development. The ability of the extra-radical mycelium (ERM) to scavenge a localised phosphate source, determined by its extraction from buried 35-μm mesh pouches, was dependent on the species of AMF tested. This work indicates that AMF from different genera have unique patterns of mycelial development when forming mycorrhizas with tropical hosts in the presence of a localised phosphate source. AMF also appear to have different mechanisms for the control of P transfer, within the mycelium, to the host. The significance of the architecture of the ERM is discussed as well as the localisation of ALP in the IRM in determining the efficiency of AMF in terms of P accumulation in planta and subsequent growth of plants. Accepted: 19 August 1998     

A survey of root pressures in vines of a tropical lowland forest

Pre-dawn xylem pressures were measured with bubble manometers attached near the stem bases of 32 species of vines on Barro Colorado Island, Panama, to determine if pressures were sufficient to allow for possible refilling of embolized vessels. Of 29 dicotyledonous species 26 exhibited only negative xylem pressures, even pre-dawn during the wet season. In contrast, three members of the Dilleniaceae exhibited positive pre-dawn xylem pressures, with a maximum of 64 kPa in Doliocarpusmajor. A pressure of 64 kPa is sufficient to push water to a height of 6.4 m against gravity, but the specimens reached heights of 18 m. Thus, in all 29 dicotyledons examined, the xylem pressures were not sufficient to refill embolized vessels in the upper stems. In contrast, two of the smaller, non-dicotyledonous vines, the climbing fern Lygodiumvenustrum and the viny bamboo Rhipidocladumracemiflorum, had xylem pressures sufficient to push water to the apex of the plants. Therefore, a root pressure mechanism to reverse embolisms in stem xylem could apply to some but not to most of the climbing plants that were studied. Received: 18 March 1996 / Accepted: 24 October 1996     

Actin coronas in normal and indeterminate gametophyte1 embryo sacs of maize

 The actin cytoskeletal organization and nuclear behavior of normal and indeterminate gametophyte1 (ig1) embryo sacs of maize were examined during fertilization. After pollination, during degeneration of one of the synergids and before arrival of the pollen tube, the cytoskeletal elements undergo dramatic changes including formation of the actin coronas at the chalazal end of the degenerating synergid and at the interface between the egg cell and central cell. The actin coronas are present only for a limited period of time and their presence is coordinated with pollen tube arrival and fusion of the gametes; they disappear before the zygote divides. This allows us to estimate the frequency of fertilized ovules along the ear. Up to 88% of the ovules on an ear contain actin coronas in the embryo sacs when observed 16–19 h after pollination, indicating the high frequency of fertilizing kernels along the ear at this stage. In the ig embryo sacs, two or more degenerated synergids containing actin coronas at their chalazal ends receive multiple pollen tubes for gametic fusion and can consequently give rise to twin or polyembryos. These findings with the monocot maize are consistent with previous reports on the dicots Plumbago and Nicotiana, suggesting that the formation of actin coronas in the embryo sac during fertilization is a universal phenomenon in angiosperms and is part of a mechanism of interaction between gametic signaling and actin cytoskeleton behavior which appears to precisely position and facilitate the access of male gametes to the egg cell and central cell for fusion. Received: 15 May 1998 / Revision accepted: 17 August 1998     

Transformation of a Texas cotton cultivar by using Agrobacterium and the shoot apex

 Transgenic cotton (Gossypium hirsutum L.) plants of a Texas cultivar CUBQHRPIS were obtained using Agrobacterium-mediated transformation coupled with the use of shoot-apex explants. After inoculation with A. tumefaciens strain LBA 4404 containing the pBI121 plasmid, regeneration of primary plants was carried out in a medium containing kanamycin (100  mg l^-1). Progeny obtained by selfing were germinated in the greenhouse and selected for expression of the T-DNA marker gene encoding neomycin phospho-transferase II (NPTII) by painting kanamycin (2%) on the leaves. Plants that survived the leaf painting were analyzed by DNA blots. Evidence for integration of the transgene (GUS) was observed in two successive generations from the regenerants (T₀). The transformed plants appeared to have more than one copy of the T-DNA. Received: 1 June 1998 / Accepted: 28 July 1998