Molecular analysis of introgressive breeding in coffee (Coffea arabica L.)

Nineteen arabica coffee introgression lines (BC₁F₄) and two accessions derived from a spontaneous interspecific cross (i.e. Timor Hybrid) between Coffea arabica (2n=4x=44) and C. canephora (2n=2x=22) were analysed for the introgression of C. canephora genetic material. The Timor Hybrid-derived genotypes were evaluated by AFLP, using 42 different primer combinations, and compared to 23 accessions of C. arabica and 8 accessions of C. canephora. A total of 1062 polymorphic fragments were scored among the 52 accessions analysed. One hundred and seventy-eight markers consisting of 109 additional bands (i.e. introgressed markers) and 69 missing bands distinguished the group composed of the Timor Hybrid-derived genotypes from the accessions of C. arabica. AFLP therefore seemed to be an extremely efficient technique for DNA marker generation in coffee as well as for the detection of introgression in C. arabica. The genetic diversity observed in the Timor Hybrid-derived genotypes appeared to be approximately double that in C. arabica. Although representing only a small proportion of the genetic diversity available in C. canephora, the Timor Hybrid obviously constitutes a considerable source of genetic diversity for arabica breeding. Analysis of genetic relationships among the Timor Hybrid-derived genotypes suggested that introgression was not restricted to chromosome substitution but also involved chromosome recombinations. Furthermore, the Timor Hybrid-derived genotypes varied considerably in the number of AFLP markers attributable to introgression. In this way, the introgressed markers identified in the analysed arabica coffee introgressed genotypes were estimated to represent from 9% to 29% of the C. canephora genome. Nevertheless, the amount of alien genetic material in the introgression arabica lines remains substantial and should justify the development of adapted breeding strategies. Received: 2 February 1999 / Accepted: 12 May 1999     

Effects of Pseudomonas fluorescens DF57 on growth and P uptake of two arbuscular mycorrhizal fungi in symbiosis with cucumber

 The effect of Pseudomonas fluorescens DF57 on growth and P uptake of two arbuscular mycorrhizal (AM) fungi in symbiosis with cucumber plants was studied in compartmentalised growth systems. Hyphae of Glomus intraradices Schenck & Smith (BEG87) or G. caledonium (Nicol. & Gerd.) Trappe & Gerdeman (BEG15) grew into lateral root-free compartments. Non-mycorrhizal plants served as control. The soil in half of the growth units of each mycorrhizal treatment was inoculated with P. fluorescens DF57. P. fluorescens DF57 enhanced hyphal length density of one of the AM fungi, G. caledonium, but this was not reflected in a higher hyphal transport of P from the root-free soil to the plant. The total P content was higher in plants grown in symbiosis with G. intraradices than in plants in the other treatments. G. caledonium and P. fluorescens DF57 had a synergistic effect in that total P content in plants inoculated with G. caledonium was higher in the presence than in the absence of P. fluorescens DF57. Accepted: 7 January 1999     

Effects of arbuscular mycorrhizal fungi and drought stress on growth and nutrient uptake of two wheat genotypes differing in drought resistance

 The effects of an arbuscular mycorrhizal (AM) fungus and drought stress on the growth, phosphorus, and micronutrient uptake of two wheat genotypes exhibiting differences in drought resistance were investigated. Plants were grown on a low P (4 mg kg^–1 soil) silty clay (Typic Xerochrept) soil-sand mix. Mycorrhizal infection was higher under well-watered than under dry soil conditions and the drought-resistant genotype CR057 had a higher mycorrhizal colonization than the drought-sensitive genotype CR006. Total and root dry matter yields and total root length were higher in mycorrhizal than in nonmycorrhizal plants of both genotypes. CR057 had higher total dry matter but not root dry matter than CR006 plants. The enhancement in total dry matter due to AM inoculation was 42 and 39% under well-watered and 35 and 45% under water-stressed for CR057 and and CR006, respectively. For both genotypes, the contents of P, Zn, Cu, Mn, and Fe were higher in mycorrhizal than in nonmycorrhizal plants and higher under well-watered than under dry soil conditions. The enhancement of P, Zn, Cu, Mn, and Fe uptake due to AM inoculation was more pronounced in CR006 than in CR057, particularly under water-stressed conditions. Thus CR006 benefitted from AM infection more than the CR057 under dry soil conditions, despite the fact that CR057 roots were highly infected. It appears that CR006 is more dependent on AM symbiosis than CR057. Accepted: 12 February 1997     

Arbuscular mycorrhizal inoculation and superphosphate application influence plant development and yield of coffee in Brazil

 This paper reports a 6-year field study of the effects of mycorrhizal pre-colonization of coffee seedlings on initial crop development and coffee bean yield in a low-fertility Oxisol amended with superphosphate (P) at planting. The experiment included five P rates (0, 20, 40, 80 and 160 g plant^–1 P₂O₅) combined with seven fungal treatments [non-mycorrhizal control, pre-colonization with a mix of Glomus clarum and Gigaspora margarita (CM) and with five isolates of Glomus etunicatum]. Inoculated and non-inoculated outplants were raised under glasshouse conditions, transplanted into the field in January 1989 and monitored until July 1995. Plant height and stem diameter were greatly enhanced by P application and were higher in mycorrhizal seedlings than in controls up to 19 months after transplanting (MAT) but were not different at 26 MAT. Inoculation effects on tree canopy diameter were significant up to 26 MAT, at which time mycorrhizal colonization was high (43–55%), but did not differ amongst plants, regardless of whether or not the plants had been pre-colonized at the nursery stage. Root colonization and spore number in the soil were reduced by high P rates at 26 MAT. The first bean yield (1991) was highly enhanced by P and all pre-colonization treatments (38% increment over control) and these factors showed a significant interaction. Three isolates of G. etunicatum showed yield enhancements above 50%. The P rate for maximal yield was 207 g plant^–1 P₂O₅ for non-pre-colonized and approximately 100 g plant^–1 for pre-colonized plants. For this harvest, the mycorrhizal biofertilizer effect was equal to 254 kg ha^–1 P₂O₅. In subsequent years, pre-colonization effects were reduced and inconsistent. In 1992, 1993 and 1995, yield was affected by P but not by mycorrhizal inoculation. In 1994 there was a P versus mycorrhiza interaction and CM and G. etunicatum-Var gave higher yields than non-precolonized plants. Considering accumulated yield for this 5-year period, P application resulted in high yield increment in all treatments, whereas pre-colonization effects were extremely diminished. However, despite inconsistency amongst mycorrhizal treatments, pre-colonization effects were detected at the fifth harvest in some fungal treatments. Based on the total yield of five harvests, maximal productivity was achieved with CM at 20 g plant^–1 P₂O₅ and with CM and G. etunicatum-Var at the highest P rate. Diminishing mycorrhizal effects over time are related to colonization of non-precolonized seedlings by the indigenous fungi and to the reduced external P requirement of the mature crop. If adequate phosphorus is applied at planting, pre-colonization of outplants with selected arbuscular mycorrhizal fungi enhances early crop development and productivity of coffee in low-fertility soils of Brazil. Accepted: 3 October 1997     

Conservation and storage of a haploid cucumber (Cucumis sativus L.) collection under in vitro conditions

 Over 3 consecutive years (1992–1994), a collection of cucumber haploids was obtained from three different lines and one hybrid. Attempts were made to maintain and store a subcollection of these haploids for 3 years. Cucumber haploids appeared to be stable when cultured in vitro. There were no instances of spontaneous doubling and only one morphologically changed plant. During the first year of storage, between 30% and 80% the clones were lost, due to disturbances in plant development, increased levels of endogenous bacteria, and physiological changes resulting in continuous flowering. After 2 years of storage haploids showed reduced vigour. Therefore, plants were regenerated directly from primordial leaf microexplants. Haploid plants were obtained from nearly all of the previous haploid plants. The rejuvenated haploids possessed the same ploidy level and morphological traits as the old collection. The only new characteristic was faster vegetative growth. Received: 17 March 1998 / Revision received: 14 April 1999 / Accepted: 10 May 1999     

The influence of ammonium nitrate on the root growth and ericoid mycorrhizal colonization of Calluna vulgaris (L.) Hull from a Danish heathland

Conversion of European heathlands to grassland has been reported as a response to increased nutrient availability, especially of nitrogen; a direct effect upon mycorrhizal colonization has been proposed as an likely explanation.This hypothesis was tested in a random block experiment with four blocks and four replicates on a Danish inland heath, Hjelm Hede. Ammonium nitrate was applied (0, 35, 50 and 70 kg N ha^–1 year^–1) to a stand of Calluna vulgaris (L.) Hull four times annually for 2 years. Calluna roots were sampled on four occasions in the 2nd year of the nitrogen treatment. The extent of ericoid mycorrhizal colonization was determined by direct observation of the roots using a line-intersection method. The nitrogen content of the current-year shoots of Calluna increased when they were treated with nitrogen. Nitrogen fertilization had no significant effects on ericoid mycorrhizal colonization of Calluna nor on root biomass. The seasonal variation in mycorrhizal colonization of the Calluna roots was highly significant. The spatial variability of mycorrhizal colonization, both in replicated plots and in the two contrasted soil horizons – the mor layer and the bleached sand – within the plots, were considerable. I conclude that heather decline under enhanced nitrogen input is unlikely to be caused by a direct impact on the ericoid mycorrhizae of Calluna. Received: 1 December 1998 / Accepted: 7 December 1999     

Effect of root exudate fractions from P-deficient and P-sufficient onion plants on root colonisation by the arbuscular mycorrhizal fungus Gigaspora margarita

 The effect of root exudates from P-deficient onion on root colonisation by an arbuscular mycorrhizal fungus was examined. Onions (Allium cepa L.) were grown in solution culture at phosphorus concentrations of 0 (P0) and 2 (P2) mg P l^–1. Root exudates were collected and fractionated with Amberlite XAD-4 resin to give EtOH and water soluble fractions. Onions inoculated with the arbuscular mycorrhizal fungus Gigaspora margarita Becker & Hall were grown with or without (control) root exudates and exudate fractions in a growth chamber. After 24 days, arbuscular mycorrhiza levels and appressoria formation had increased in plants treated with P0-root exudate or the P0-EtOH fraction when compared to corresponding P2 treatments or control plants. P0 and P2 water-soluble fractions did not significantly affect either aspect of fungal development. These results suggest that hydrophobic compounds found in root exudates from P-deficient onion increase appressorium formation and, therefore, enhance mycorrhiza development. Accepted: 2 June 1998     

Comparison of somatic embryogenesis-derived coffee (Coffea arabica L.) plantlets regenerated in vitro or ex vitro: morphological,mineral and water characteristics

Coffea arabica L. plantlets obtained ex vitro after sowing somatic embryos produced in a bioreactor in horticultural substrate were compared with those obtained in vitro from the same embryo population under conventional culturing conditions on semi-solid media. The intensity and quality of aerial and root system development were compared. Shoot emergence was more efficient in vitro but rooting frequencies were low. In contrast, all ex vitro-regenerated embryos rooted. The cotyledon area of mature embryos produced in a bioreactor positively affected plantlet development when regeneration was carried out ex vitro. Embryos with an intermediate cotyledon area (0.86 cm2) had the highest rates of plant conversion ex vitro (63%), and also resulted in vigorous plantlets. Mortality was higher in nursery conditions, but better plant development was obtained. The quality of plantlets produced under ex vitro conditions was reflected in better growth of the aerial and root systems, and also by similar morphological, mineral and water status characteristics to seedlings. Unlike roots formed on semi-solid media, those produced in soil were branched, fine (30-50% had a diameter of less than 0-5 mm) and they bore root hairs. Leaves of plantlets regenerated ex vitro had a histological structure similar to that of seedling leaves, and a lower stomatal density (100 vs. 233 mm-2). Moreover, they were more turgid, as indicated by higher pressure potential (psiP) (0.91 s. 0.30 MPa) and relative water content values (97 vs. 93%). Furthermore, under in vitro conditions, leaves had larger stomata which were abnormally round and raised. Direct sowing of germinated somatic embryos resulted in the rapid production of vigorous plantlets under ex vitro conditions, whilst removing the need for problematical and costly conventional acclimatization procedures.     

In vitro double fertilization in Nicotiana tabacum (L.): fusion behavior and gamete interaction traced by video-enhanced microscopy

In vitro double fertilization in tobacco was carried out with attention to fusion behavior and gamete interaction. Structural and cytological events indicating possible reaction to the fusion of sperm-egg and especially sperm-central cell were recorded by video-enhanced microscopy. Generative cells were fused with the egg cell or central cell as a control system to better understand gamete interaction. As early as adherence of the male cell, the female cell showed response by means of cytoplasm strand formation. After gamete fusion, cytoplasm activation in the egg cell was observed as long distance movement of organelles. In fertilized central cells, however, fusion did not result in notable cytological change within 30 min. Male nuclear movement recorded in the female cell illustrated two different patterns of movement which showed similarity to organelle movement. The dynamics of male and female nuclear fusion after in vitro fertilization was also recorded in the central cell. It revealed that the fusion process requires only a few seconds and is similar to that of gamete fusion in vitro. This may offer a new clue for understanding how female and male nuclei attract, adhere and finally fuse each other. Received: 13 October 1999 / Revision accepted: 6 December 1999     

Arbuscular mycorrhizal symbiosis and nonhydraulic signaling of soil drying in Vigna unguiculata (L.) Walp.

 We examined the influence of Glomus intraradices on nonhydraulic signaling of soil drying, in a drought-avoiding plant having stomates that are extremely sensitive to changes in soil moisture. Cowpea [Vigna un guiculata (L.) Walp. 'White Acre'] seedlings were grown in a greenhouse with root systems split between two pots. The 2×3×2 experimental design included two levels of mycorrhizal colonization (presence or absence of Glomus intraradices Schenck & Smith UT143), three levels of phosphorus fertilization within each mycorrhizal treatment and two levels of water (both pots watered or one pot watered, one pot allowed to dry). Stomatal conductance was mostly similar in fully watered mycorrhizal and nonmycorrhizal controls. However, g _s of half-dried, nonmycorrhizal plants was reduced on fewer days and to a lesser extent than g _s of half-dried, mycorrhizal plants, perhaps related to quicker soil drying in mycorrhizal pots. The partial soil drying treatment had little effect on leaf relative water content or osmotic potential, indicating that declines in g _s and leaf growth were induced by some nonhydraulic factor. Leaf growth was inhibited only in nonmycorrhizal plants, evidently due to a difference in phosphorus nutrition between mycorrhizal and nonmycorrhizal plants. The mycorrhizal effect on g _s was not associated with phosphorus nutrition. Inhibition of g _s was directly related to extent of soil drying, while inhibition of leaf growth was inversely related to extent of soil drying. Accepted: 4 August 1995     

Recurrent production of plants of black plum, Syzygium cuminii (L.) Skeels, a myrtaceous fruit tree, from in vitro cultured seedling explants

 The epicotyl segments bearing scaly leave(s), excised from in vitro grown seedlings of Syzygium cuminii, produced multiple shoots when cultivated on Murashige and Skoog's (MS, 1962) medium supplemented with different concentrations of BA (0–2 mg l^–1). The optimum response was recorded on the medium containing 1 mg l^–1 BA. An average of 8.6 shoots per explant were produced 60 days after inoculation, following transfer to fresh medium after 30 days. The shoots were excised, and the residual explants were transferred to fresh medium, where they again developed shoots. Up to five such passages resulted in the production of shoots from the repeatedly subcultured original explants. However, during the fifth passage, organogenic response was negligible and the explants turned brown thereafter. Following repeated harvesting of shoots and subculture of the residual explants, an average of 29 shoots per explant was obtained. The in vitro developed shoots produced roots when transferred to Knop's medium supplemented with 2% sucrose and 1 mg l^–1 IAA. The developed plantlets were planted in soil and transferred to fields after an acclimatization period of 7–8 months. These plants have been thriving well for more than 3 years. The nodal explants excised from in vitro developed shoots and plants also exhibited a similar response when cultured on MS+1 mg l^–1 BA. Thus, a protocol has been developed to raise plants of S. cuminii at any time of the year. Received: 1 December 1998 / Revision received: 1 July 1999 · Accepted: 12 July 1999     

In vitro double fertilization in Nicotiana tabacum (L.): polygamy compared with selected single pair somatic protoplast and chloroplast fusions

In vitro polygamy was studied mainly by using isolated sperm and central cells of tobacco in order to elucidate the mechanism that might be involved in preventing in vivo polygamy. In 17.5% 4000 M.W. polyethylene glycol, only when two sperm cells were made close enough to each other and adhered to a female cell simultaneously was polygamy possible. If one sperm cell fused with the egg or central cell, within 30 min another sperm cell could not fuse with the same egg or central cell. Similar phenomena were found in selected single somatic cell fusion. When more than two protoplasts adhered to each other simultaneously, fusion was always successful; after two protoplasts fused, within 30 min the fusion products could not fuse with another protoplast under the same conditions. This comparative study revealed this characteristic to be shared by both sexual and somatic cell fusion. However, after cytoplasm reorganization was complete in the fusion product, it was possible for the fusion product to fuse with the third protoplast. This indicates that the obstruction to additional fusion was present only during a certain period after the preceding fusion under certain condition. The possible reason for the effect is discussed. Received: 7 March 2000 / Revision accepted: 15 June 2000     

Organic nitrogen use by salal ericoid mycorrhizal fungi from northern Vancouver Island and impacts on growth in vitro of Gaultheria shallon

 Salal (Gaultheria shallon) recovers quickly from rhizomes after clear-cut timber harvesting and dominates clearcuts of Tsuga heterophylla and Thuja plicata forests. Thus it contributes to considerable problems in regeneration of these sites in coastal British Columbia, Canada. Based on what is known about other ericaceous plants, we speculated that mycorrhizal fungi of salal play a vital role in the growth and dominance of salal by providing access to organic nitrogen. In this study, the ability of four species of fungi isolated from salal to use different forms of organic nitrogen was tested in pure culture and in association with salal. The organic forms of nitrogen applied were glutamine (an amino acid), glutathione (a peptide), and bovine serum albumin (BSA, a protein). The fungi tested were Oidiodendron maius, Acremonium strictum, and two nonsporulating fungi. Inoculated plants always grew better than noninoculated plants regardless of nitrogen source. Glutamine was used as readily as ammonium nitrogen by all four fungi and the mycorrhizal plants of salal colonized by those fungi. There was considerable variation between fungus species or the plants inoculated with those fungi in using glutathione and BSA. Salal inoculated with O. maius grew better on glutathione than BSA, while A. strictum and unknown 1 produced significantly greater yields of salal on BSA. Colonization rates of salal by all four fungi was higher on glutathione or BSA than on ammonium or glutamine. Accepted: 19 June 1999     

Effects of soil sterilization,inoculation with vesicular-arbuscular mycorrhizal fungi and cropping history on peanut (Arachis hypogaea L.) growth in an oxisol from subtropical Australia

The effects of soil sterilization and inoculation with vesicular-arbuscular mycorrhizal fungi (VAMF) on VAMF colonization, nutrition and growth of peanut plants (cv. Virginia Bunch) were investigated in an Oxisol soil in pots in the glasshouse. Sterilization, nutrient and inoculation treatments were applied to soil with a history of either continuous summer cropping (>50 years) or continuous grass pasture. Root colonization by VAMF was strongly affected by sterilization of soil from both cropping histories. Irradiation and aerated steam treatments virtually eliminated VAMF colonization; methyl bromide+chloropicrin caused an early reduction in colonization which was no longer visible twenty-one days after flowering; and DD+methyl isothiocyanate caused no significant reduction. Despite earlier differences in VAMF colonization of roots, plant growth responses were not recorded until after flowering. Plant growth 21 days after flowering was strongly correlated with VAMF colonization at first flower and at 21 days after first flower. Effective soil sterilization caused significant reductions in plant growth which were eliminated by addition of VAMF inoculum. Growth reductions in the absence of VAMF could be directly related to severe P deficiency, which was reflected in P uptake as early as first flower; Zn uptake was also reduced. Although VAMF colonization was slightly higher in grassland than in continuously cropped soil, growth responses to VAMF inoculation were not observed in unsterilized soil regardless of cropping history, indicating a general adequacy of resident VAMF populations. Plants grew better in untreated cropped soil than in grassland soil although growth was poor in both after effective soil sterilization. These differences could be related to the higher level of available P in the cropped soil and the inability of plants to access this P in the absence of VAMF. Regular applications of N-free nutrient solution had no effect on plant growth even in effectively sterilized soil, suggesting a threshold response to P in the absence of VAMF in this soil.     

Effects of vesicular-arbuscular mycorrhizae on growth and phosphorus and zinc nutrition of peanut (Arachis hypogaea L.) in an Oxisol from subtropical Australia

Peanut plants (cv. Shulamit) were grown in an Oxisol soil in pots in the glasshouse to assess effects of soil sterilization and inoculation with spores of vesicular-arbuscular mycorrhizal fungi (VAMF) on the response to five rates of phosphorus (0 to 240 kg P ha^–1) and two rates of zinc (0 and 10 kg Zn ha^–1) fertilizers.Both P and Zn nutrition were affected by VAMF activity but the dominant role of VAMF in this soil type was in uptake of P. In the absence of VAMF there was a clear threshold level of P application (60 kg P ha^–1) below which plants grew poorly, which resulted in a sigmoidal response of dry matter to applied P. The maximum response was not fully defined because dry matter production continued to increase up to 240 kg P ha^–1. Tissue P concentration of non-mycorrhizal plants increased linearly with P rate and was always significantly less than that in mycorrhizal plants.Mycorrhizal plants responded without threshold to increasing P rate, attaining maximum dry matter at 120 kg P ha^–1 in inoculated sterilized soil and at 30 kg P ha^–1 in nonsterile soil. These differences in maximal P rates and in the greater dry matter produced in sterile soil at high P rates were attributed to the negative effects of the root-knot nematodeMeloidogyne hapla in nonsterile soil.Plant weight did not respond to zinc fertilizer but tissue Zn concentration increased with applied Zn. Tissue Zn concentration and uptake were increased by VAMF.     

Effects of a vesicular-arbuscular mycorrhizal fungus and other soil microorganisms on growth,mineral nutrient acquisition and root exudation of soil-grown maize plants

Maize (Zea mays L. cv. Alize) plants were grown in a calcareous soil in pots divided by 30-m nylon nets into three compartments, the central one for root growth and the outer ones for hyphal growth. Sterle soil was inoculated with either (1) rhizosphere microorganisms other than vesicular-arbuscular mycorrhizal (VAM) fungi, (2) rhizosphere microorganisms together with a VAM fungus [Glomus mosseae (Nicol. and Gerd.) Gerdemann and Trappel], or (3) with a gamma-irradiated inoculum as control. Plants were grown under controlled-climate conditions and harvested after 3 or 6 weeks. VAM plants had higher shootroot ratios than non-VAM plants. After 6 weeks, the concentrations of P, Zn and Cu in roots and shoots had significantly increased with VAM colonization, whereas Mn concentrations had significantly decreased. Root exudates were collected on agar sheets placed on the interface between root and hyphal compartments. Six-week-old VAM and non-VAM plants had similar root exudate compositions of 72–73% reducing sugars, 17–18% phenolics, 7% organic acids and 3% amino acids. In another experiment in which root exudates were collected on agar sheets with or without antibiotics, the amounts of amino acids and carbohydrates recovered were similar in VAM and non-VAM plants. However, threeto sixfold higher amounts of carbohydrates, amino acids and phenolics were recovered when antibiotics were added to the agar sheets. Thus, the high microbial activity in the rhizosphere and on the rhizoplane limits the exudates recovered from roots.     

In vitro pollen tube growth and penetration of female gametophyte in Douglas fir (Pseudotsuga menziesii)

 Pollen tube and female gametophyte interactions in Douglas fir (Pseudotsuga menziesii) were examined in vitro. Formation of pollen tubes in Douglas fir occurred on a modified Murashige and Skoog medium in which concentrations of H₃BO₃ and Ca(NO₃)₂ were altered and supplemented with sucrose and polyethylene glycol. Addition of 100 μg/ml H₃BO₃ and 300 μg/ml Ca(NO₃)₂ resulted in optimum pollen viability. Lack of H₃BO₃ inhibited pollen tube formation. Addition of H₃BO₃ and Ca(NO₃)₂ significantly increased pollen tube formation within one week in culture. Using a medium supplemented with mannitol, viability of Douglas fir pollen can be sustained for 7 weeks in culture, about the same length of time as in vivo. However, pollen tubes are not formed. This suggests that the factors responsible for tube formation reside in the external environment of the pollen. Culture of female gametophytes to examine egg viability and longevity had not been done previously. We found that egg viability in culture is short-lived, and therefore the window to study and manipulate events of fertilization in Douglas fir is very limited. In spite of this, about 7% of the female gametophytes that were co-cultured became penetrated by pollen tubes. In vitro archegonial penetration has been repeatedly achieved, but pollen tubes also penetrated other parts of the female gametophytes. Pollen tubes also penetrated non-viable eggs. Most female gametophytes were not penetrated because of pollen tube branching and swelling, failure of tubes to orient towards the female gametophytes, or premature pollen tube death due to plasmolysis. This report outlines the first attempt towards in vitro fertilization in conifers. Received: 13 March 1997 / Revision accepted: 6 June 1997     

Effects of liming and mycorrhizal colonization on soil phosphate depletion and phosphate uptake by maize (Zea mays L.) and soybean (Glycine max L.) grown in two tropical acid soils

The effects of liming and inoculation with the arbuscular mycorrhizal fungus, Glomus intraradices Schenck and Smith on the uptake of phosphate (P) by maize (Zea mays L.) and soybean (Glycine max [L.] Merr.) and on depletion of inorganic phosphate fractions in rhizosphere soil (Al-P, Fe-P, and Ca-P) were studied in flat plastic containers using two acid soils, an Oxisol and an Ultisol, from Indonesia. The bulk soil pH was adjusted in both soils to 4.7, 5.6, and 6.4 by liming with different amounts of CaCO₃.In both soils, liming increased shoot dry weight, total root length, and mycorrhizal colonization of roots in the two plant species. Mycorrhizal inoculation significantly increased root dry weight in some cases, but much more markedly increased shoot dry weight and P concentration in shoot and roots, and also the calculated P uptake per unit root length. In the rhizosphere soil of mycorrhizal and non-mycorrhizal plants, the depletion of Al-P, Fe-P, and Ca-P depended in some cases on the soil pH. At all pH levels, the extent of P depletion in the rhizosphere soil was greater in mycorrhizal than in non-mycorrhizal plants. Despite these quantitative differences in exploitation of soil P, mycorrhizal roots used the same inorganic P sources as non-mycorrhizal roots. These results do not suggest that mycorrhizal roots have specific properties for P solubilization. Rather, the efficient P uptake from soil solution by the roots determines the effectiveness of the use of the different soil P sources. The results indicate also that both liming and mycorrhizal colonization are important for enhancing P uptake and plant growth in tropical acid soils.     

Effects of carbon dioxide enrichment on response of mycorrhizal pitch pine (Pinus rigida) to aluminum: growth and mineral nutrition

 Carbon dioxide enrichment may increase the Al tolerance of trees by increasing root growth, root exudation and/or mycorrhizal colonization. The effect of elevated CO₂ on the response of mycorrhizal pitch pine (Pinus rigida Mill.) seedlings to Al was determined in two experiments with different levels of nutrients, 0.1- or 0.2-strength Clark solution. During each experiment, seedlings inoculated with the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker & Couch were grown 13 weeks in sand irrigated with nutrient solution (pH 3.8) containing 0, 6.25, 12.5, or 25 mg/l Al (0, 232, 463, or 927 μM Al) in growth chambers fumigated with 350 (ambient) or 700 (elevated) μl/l CO₂. At ambient CO₂, in the absence of Al, mean total dry weights (DW) of seedlings at the high nutrient level were 164% higher than those at the low level. Total DW at elevated CO₂, in the absence of Al, was significantly greater than that in ambient CO₂ at the low (+34%) and high (+16%) nutrient levels. Root and shoot DW at both nutrient levels decreased with increasing Al concentrations with Al reducing root growth more than shoot growth. Although visible symptoms of Al toxicity in roots and needles were reduced by CO₂ enrichment, there were no significant CO₂ × Al interactions for shoot or root DW. The percentage of seedling roots that became mycorrhizal was negatively related to nutrient level and was greater at elevated than at ambient CO₂ levels. Generally, elevated CO₂ had little effect on concentration of mineral nutrients in roots and needles. Aluminum reduced concentrations of most nutrients by inhibiting uptake. Received: 18 June 1997 / Accepted: 8 December 1997