Growth and Nodulation Responses of Rhizobium meliloti to Water Stress Induced by Permeating and Nonpermeating Solutes

Isolates of Rhizobium meliloti, representing antigenically distinct indigenous serogroups 31 and 17, were grown in yeast extract-mannitol broth (YEM) containing NaCl or polyethylene glycol (PEG) to provide external water potentials ranging from −0.15 to −1.5 MPa. Several differences were found between representatives of the two groups in their abilities to adapt to water stress induced by the nonpermeating solute PEG. At potentials below −0.5 MPa, strain 31 had a lower specific growth rate than strain 17 and an irregular cell morphology. In contrast, neither growth nor cell morphology of either strain was affected significantly over the same range of water potentials created by a permeating solute, NaCl. Despite the superior growth of strain 17 at the low water potentials imposed by PEG, upshock of water-stressed cells (−1.0 MPa; PEG) into normal YEM (−0.15 MPa) resulted in a faster recovery of growth by strain 31 than by strain 17. Different responses of the two strains to a water potential increase were also revealed in nodulation studies. Strain 31 required significantly fewer days to nodulate alfalfa than strain 17 did when the strains were transferred from YEM with PEG at −1.0 MPa onto the roots of alfalfa seedlings in plant growth medium (−0.1 MPa). The addition of supplemental calcium (0.1 mM) to growth medium with PEG (−1.0 MPa) reduced the differences between strains in their responses to water stress. The severe growth restriction and morphological abnormalities shown by strain 31 were corrected, and the prolonged recovery time shown by water-stressed cells (−1.0 MPa; PEG) of strain 17 upon transfer to normal YEM was shortened. The latter strain also nodulated earlier and more rapidly after growth in PEG medium at −1.0 MPa in the presence of supplemental calcium ions. These results indicate that the efficacy of osmoregulation can vary among strains of the same species and that the mechanism of osmoregulation may differ depending on the nature of the water stress.     

Influence of drought on competition between selected Rhizobium meliloti strains and naturalized soil rhizobia in alfalfa

Drought is an important environmental factor that can affect rhizobial competition and N₂ fixation. Three alfalfa (Medicago sativa L. and M. falcata L.) accessions were grown in pots containing soil from an irrigated (Soil 1) and a dryland (Soil 2) alfalfa field in northern Utah, USA. Mutants of three strains of Rhizobium meliloti Dang. from Pakistan (UL 136, UL 210, and UL 222) and a commercial rhizobial strain 102F51a were developed with various levels of resistance to streptomycin. Seeds inoculated with these individual streptomycin-resistant mutants were sown in the two soils containing naturalized rhizobial populations. Soils in the pots were maintained at −0.03, −0.5, and −1.0 MPa. After 10 weeks, plants were harvested and nodule isolates were cultured on agar medium with and without streptomycin to determine nodule occupancy (proportion of the nodules occupied by introduced rhizobial strains). Number of nodules, nodule occupancy, total plant dry weight, and shoot N were higher for Soil 1 than Soil 2. Number of nodules, plant dry weight, and shoot N decreased as drought increased from −0.03 to −1.0 MPa in the three alfalfa accessions. Rhizobial strains UL 136 and UL 222 were competitive with naturalized alfalfa rhizobia and were effective at symbiotic N₂ fixation under drought. These results suggest that nodulation, growth, and N₂ fixation in alfalfa can be improved by inoculation with competitive and drought-tolerant rhizobia and may be one economically feasible way to increase alfalfa production in water-limited environments. Joint contribution from USDA-ARS and the Utah Agric. Exp. Sta., Utah State Univ., Logan, UT 84322-4810, USA. Journal Paper No. 4931. Joint contribution from USDA-ARS and the Utah Agric. Exp. Sta., Utah State Univ., Logan, UT 84322-4810, USA. Journal Paper No. 4931.     

High Salt and High pH Tolerance of New Isolated Rhizobium etli Strains from Egyptian Soils

Saline and alkaline soils are major problems contributing to the low productivity of common bean (Phaseolus vulgaris) in arid and semi-arid regions such as Egypt. Therefore our study was directed toward selecting strains more tolerant to these environmental stresses. Among seven Rhizobium etli strains isolated from Egyptian soils, we found a high degree of diversity. Strains EBRI 21 and EBRI 26 are highly tolerant to a salt concentration up to 4% NaCl. A positive correlation was found between the salt tolerance and the adaptation to alkaline pH (9). Strains EBRI 2 and EBRI 26 were adapted to elevated temperatures (42°C). The minimum level of low pH for the majority of Rhizobium etli strains from Egypt was pH 4.7 while the Colombian strain Rhizobium tropici CIAT 899 survived well at pH 4. At 0.4% NaCl, the symbiotic efficiency of the salt-tolerant strain EBRI 26 was superior in cultivar Giza 6 compared with the salt-sensitive strain EBRI 2 (18.2 compared with 13.9 nM C₂H₄ h^–1 mg^–1 nodule fresh weight). In the bean cultivar Saxa, nitrogen fixation was much more affected by high salt concentration (0.4% NaCl) than in the cultivar Giza 6 with both strains (3.9 and 3.8 nM C₂H₄ h^–1 mg^–1 nodule fresh weight, respectively). In general, stress of alkalinity had a less detrimental effect on nodulation and N₂ fixation than stress of salinity.     

The Effect of Different Salts of Sodium and Potassium on the Accumulation of Glycinebetaine in Atriplex Prostrata

Atriplex prostrata was grown for one month in nutrient solutions with NaCl, KCl, Na₂SO₄, and K₂SO₄ (at osmotic potentials of 0, –0.75, –1.00, and –1.50 MPa). Plants treated with K₂SO₄ had less glycinebetaine at –1.0 and –1.50 MPa than those treated with Na⁺ salts, probably due to the inhibitory effects of K⁺ on glycinebetaine accumulation.     

Salt Tolerance of In Vitro Established Salt-Tolerant Rice Plants during Further Growth in Soil

In vitro salt tolerant rice plants established by step up treatment with 0.5, 1.0, 1.5 and 2.0 % NaCl at 3-week intervals were examined to determine whether they could grow in potted paddy soil containing 0, 0.55 or 0.75 % NaCl till harvesting. All the control plants were necrotic by the 4^th week in the culture. At the 10^th week of culture, 100 % of the salt-tolerant plants subjected to 0 or 0.55 % NaCl survived, and 78 % of the plants at 0.75 % NaCl. The Na⁺ and Cl^– contents in the leaves of salt-tolerant plants grown at 0.55 and 0.75 % NaCl were about 4 times of those without NaCl. The ion contents in non-tolerant plants and seedling plants were 10 to 12 times of those in 0 % NaCl treatment. One of the hypotheses to explain the present data is that the in vitro step up salt selection induces the capability to maintain no lethal concentration of NaCl in the leaves.     

Utilization of the tricarboxylic acid cycle intermediates and symbiotic effectiveness inRhizobium meliloti

Summary The utilization of the tricarboxylic acid cycle intermediates and related compounds was studied in strains ofRhizobium meliloti having different symbiotic effectiveness. In general, the very effective (VE) strains used these compounds as sole carbon source better than the ineffective (I) strains. However, a significant different was observed between VE and I strains in their ability to use acetate or oxaloacetate for growth. In fact, at a concentration of 2 mM, 80% of the VE strains used acetate or oxaloacetate white 50% of the I strains used acetate and none was able to grow on oxaloacetate. No correlation was found between the symbiotic effectiveness of the strains and their ATP content, when grown on mannitol. The highest ATP content (9.21 nM×g protein^–1) was found in the I strain S₂₀ and the lowest (0.69 nM×g protein^–1 was found in the effective strain S₈. Numerical analysis of the patterns of utilization of the TCA cycle intermediates and related compounds indicated that the 49 strains tested formed 11 distinct groups at 86% similarity, according to Jaccard's coefficient. Several strains showed unique patterns of utilization and can be clearly identified under laboratory conditions.Contribution no.225 Station de Recherches, Agriculture Canada.     

Physiology, biochemistry and taxonomy of deep-sea nitrile metabolising Rhodococcus strains

A collection of nitrile-hydrolysing rhodococci was isolated from sediments sampled from a range of deep coastal, and abyssal and hadal trench sites in the NW Pacific Ocean, as part of our programme on the diversity of marine actinomycetes. Nitrile-hydrolysing strains were obtained by batch enrichments on nitrile substrates with or without dispersion and differential centrifugation pre-treatment of sediments, and were recovered from all of the depths sampled (approximately 1100–6500 m). Two isolates obtained from the Ryukyu (5425 m) and Japan (6475 m) Trenches, and identified as strains of Rhodococcus erythropolis,were chosen for detailed study. Both of the deep-sea isolates grew at in situ temperature (4°C), salinities (0–4% NaCl) and pressures (40–60 MPa), results that suggest, but do not prove, that they may be indigenous marine bacteria. However, the absence of culturable Thermoactinomycespoints to little or no run off of terrestrial microbiota into these particular trench sediments. Nitrile-hydrolysis by these rhodococci was catalysed by a nitrile hydratase–amidase system. The hydratase accommodated aliphatic, aromatic and dinitrile substrates, and enabled growth to occur on a much wider range of nitriles than the only other reported marine nitrile-hydrolysing R. erythropolis which was isolated from coastal sediments. Also unlike the latter strain, the nitrile hydratases of the deep-sea rhodococci were constitutive. The possession of novel growth and enzyme activities on nitriles by these deep-sea R. erythropolisstrains recommends their further development as industrial biocatalysts.     

In vitro selection for salt tolerant lines in Lycopersicon peruvianum

A salt-tolerant callus line of Lycopersicon peruvianum has been obtained by exposing the cells, in suspension cultures and then in callus, to increasing concentrations of NaCl (50–350mM). This selected line grew better than the nonselected line at all levels of NaCl. Moreover, this selected line grew better in media containing salt than in those without it. It retained its tolerance after subculture for 3 passages (3 months) on salt-free medium. The growth of the selected line in mannitol was similar to that of the nonselected line, which suggested that the superiority of the selected line under salt stress was not due to osmotic stress tolerance. The ions SO ₄ ^–– and K⁺ were highly toxic to L. peruvianum root callus, while Na⁺, Mg⁺⁺ and Cl^– were less toxic.     

Effect of salinity on growth of four strains of Rhizobium and their infectivity and effectiveness on two species of Acacia

Two Rhizobium strains (WU1001 and WU1008) were isolated from nodules of Acacia redolens growing in saline areas of south-west Australia, and two strains selected from the University of Western Australia's culture collection (WU429 isolated from A. saligna and WU433 from A. cyclops). The growth of each in buffered, yeast extract mannitol broth culture was largely unaffected by salt up to 300 mM NaCl. A slight increase in lag time occurred at concentrations of 120 mM NaCl and above, but cell number at the static phase was not affected. Each of the four Rhizobium strains tested accumulated Na⁺ but showed decreasing levels of sugar with increasing salt in the external medium. Amino acid levels also increased, in some cases by more than tenfold. However, the relative proportion of each remained fairly constant in the bacteria, irrespective of salt treatment. Only trace quantities of proline were detected and there was no increase in this amino acid with salt. Acidic amino acids (glutamate and aspartate) remained as a constant proportion.Rhizobium strains WU429, WU1001 and WU1008 produced effective nodules on both A. cyclops and A. redolens grown in sand with up to 80 mM NaCl (added in nutrient solutions free of nitrogen). Strain WU433 was highly infective on both Acacia species tested at low salt concentrations (2–40 mM NaCl), but infection was sensitive to salt levels at 120 mM NaCl and above. Nodules formed with strain WU433 were, however, ineffective on both A. redolens and on A. cyclops and showed nil or negligible rates of acetylene reduction at all salt concentrations. Strains WU429, WU1001 and WU1008 in combination with a highly salt-tolerant provenance of A. redolens formed symbioses which did not vary significantly in nodule number and mass, specific nodule activity or total N content irrespective of salt level up to 160 mM NaCl. On a more salt sensitive provenance of A. redolens and on A. cyclops the infectivity and effectivity of the Rhizobium strains tested usually decreased as the external salt concentration increased. These data are interpreted to indicate that tolerance of the legume host was the most important factor determining the success of compatible Rhizobium strains in forming effective symbioses under conditions of high soil salinity.     

Selection and breeding for salinity tolerancein vitro

Summary Selection for tolerance to NaCl inCitrus sinensis andC. aurantium has been carried out in agar and suspension cultures. Callus was subjected to culture media containing up to 0.17M NaCl for ten passages. Selected cell lines were grown for three passages on media without salt before further tests on saline media. Four stable tolerant cell lines, differing in degree of tolerance, have been selected fromC. sinensis. Four lines of similar tolerance have been selected fromC. aurantium. The stability of most lines was very satisfactory. MostC. sinensis lines grew well in media containing up to 0.2M NaCl, andC. aurantium lines in media of up to 0.15M NaCl.Embryos were regenerated in most selected cell lines fromC. sinensis and, more sporadically, fromC. aurantium. Addition of 0.5–0.6% NaCl to the media often enhanced embryogenesis. Embryos from a selected line ofC. sinensis showed higher tolerance to NaCl in the medium than comparable embryos from an unselected line.Single embryos derived from both selected and unselected cell lines ofC. sinensis were successfully cloned. A limited comparison of plantlets from one tolerant line (R14) with plantlets from unselected control lines showed better adaptation of the former to salt (0.085 to 0.12M NaCl in the medium), and a lesser degree of leaf burn symptoms.Contribution No. 1045-E, 1984 series.     

Effect of amaranth on the growth of Rhizobium and Bradyrhizobium strains

The growth rate of different strains of Bradyrhizobium and Rhizobium was studied in media containing amaranth seed meal instead of yeast extract. Results obtained in erlenmeyer flasks and stirred fermenters show that both Bradyrhizobium japonicum strains E109, E110, 5019, 587 and Rhizobium melilotistrains B36, B323, B399, Lq₂₂, Lq₄₂, Lq₅₁ and U₃₂₂, grow satisfactorily in amaranth seed meal medium. Cell count obtained for the strains tested was greater than 4 × 10¹⁰ viable cells.ml^–1. Amaranth seed meal (4 g.l^–1) is a suitable component for culture media that can be used instead of yeast extract.     

Relative importance of Na+, Cl−, and abscisic acid in NaCl induced inhibition of root growth of rice seedlings

The relative importance of endogenous abscisic acid (ABA), as well as Na⁺ and Cl^– in NaCl-induced responses related to growth in roots of rice seedlings were investigated. The increase in ammonium, proline and H₂O₂ levels, and cell wall peroxidase (POD) activity has been shown to be related to NaCl-inhibited root growth of rice seedlings. Increasing concentrations of NaCl from 50 to 150 mM progressively decreased root growth and increased both Na⁺ and Cl^–. Treatment with NaCl in the presence of 4,4-diisothiocyano-2,2-disulfonic acid (DIDS, a nonpermeating amino-reactive disulfonic acid known to inhibit the uptake of Cl^–) had less Cl^– level in roots than that in the absence of DIDS, but did not affect the levels of Na⁺, and responses related to growth in roots. Treatment with 50 mM Na-gluconate (the anion of which is not permeable to membrane) had similar Na⁺ level in roots as that with 100 mM NaCl. It was found that treatment with 50 mM Na-gluconate effected growth reduction and growth-related responses in roots in the same way as 100 mM NaCl. All these results suggest that Cl^– is not required for NaCl-induced responses in root of rice seedlings. Endogenous ABA level showed no increase in roots of rice seedlings exposed to 150 mM NaCl. It is unlikely that ABA is associated with NaCl-inhibited root growth of rice seedlings.     

Siderophore and organic acid production in root nodule bacteria

Nineteen strains of root nodule bacteria were grown under various iron regimes (0.1, 1.0 and 20 M added iron) and tested for catechol and hydroxamate siderophore production and the excretion of malate and citrate. The growth response of the strains to iron differed markedly. For 12 strains (Bradyrhizobium strains NC92B and 32H1, B. japonicum USDA110 and CB1809, B. lupini WU8, cowpea Rhizobium NGR234, Rhizobium meliloti strains U45 and CC169, Rhizobium leguminosarum bv viciae WU235 and Rhizobium leguminosarum bv trifolii strains TA1, T1 and WU95) the mean generation time showed no variation with the 200-fold increase in iron concentration. In contrast, in Bradyrhizobium strains NC921, CB756 and TAL1000, B. japonicum strain 61A76 and R. leguminosarum bv viciae MNF300 there was a 2–5 fold decrease in growth rate at low iron. R. meliloti strains WSM419 and WSM540 showed decreased growth at high iron.All strains of root nodule bacteria tested gave a positive CAS (chrome azurol S) assay for siderophore production. No catechol-type siderophores were found in any strain, and only R. leguminosarum bv trifolii T1 and bv viciae WU235 produced hydroxamate under low iron (0.1 and 1.0 M added iron).Malate was excreted by all strains grown under all iron regimes. Citrate was excreted by B. japonicum USDA110 and B. lupini WU8 in all iron concentrations, while Bradyrhizobium TAL1000, R. leguminosarum bv viciae MNF300 and B. japonicum 61A76 only produced citrate under low iron (0.1 and/or 1.0 M added iron) during the stationary phase of growth.Abbreviations CAS chrome azurol S - HDTMA hexadecyltrime-thylammonium bromide     

Effects of salt on rhizobia and bradyrhizobia: a review

Rhizobia and bradyrhizobia strains vary in their tolerance to salt-stress. Rhizobium strains (fast-growers) are more salt-tolerant than strains of Bradyrhizobium (slow-growers). However, salt-tolerance in both genera is dependent upon ionic species, pH value, temperature, carbon source and the presence of osmoprotectant solutes. The harmful effect of salts on growth of both genera can be attributed to the specific ion effect rather than the osmotic effect. The salt-tolerance of different strains of rhizobia and bradyrhizobia is not related to their ecological origin. Data for salt tolerance of 684 strains of rhizobia and bradyrhizobia were collected from many reports. Most of the reports confound the effects of salt and express the concentrations of salts in percentage (%), electrical conductivity (dS m^-1), molar concentration (m ) or osmotic pressure (MPa) regardless of their differences. All the published data were compiled and recalculated from the different expressions to their equivalent molar concentration (m ) of NaCl. A suggested classification of salt-tolerance of rhizobia and bradyrhizobia from the compiled data is presented.     

Characterization of recA genes and recA mutants of Rhizobium meliloti and Rhizobium leguminosarum biovar viciae

Summary DNA fragments carrying the recA genes of Rhizobium meliloti and Rhizobium leguminosarum biovar viciae were isolated by complementing a UV-sensitive recA ^– Escherichia coli strain. Sequence analysis revealed that the coding region of the R. meliloti recA gene consists of 1044 by coding for 348 amino acids whereas the coding region of the R. leguminosarum bv. viciae recA gene has 1053 bp specifying 351 amino acids. The R. meliloti and R. leguminosarum bv. viciae recA genes show 84.8% homology at the DNA sequence level and of 90.1% at the amino acid sequence level. recA ^– mutant strains of both Rhizobium species were constructed by inserting a gentamicin resistance cassette into the respective recA gene. The resulting recA mutants exhibited an increased sensitivity to UV irradiation, were impaired in their ability to perform homologous recombination and showed a slightly reduced growth rate when compared with the respective wild-type strains. The Rhizobium recA strains did not have altered symbiotic nitrogen fixation capacity. Therefore, they represent ideal candidates for release experiments with impaired strains.The accession numbers: X59956 R. LEGUMINOSARUM REC A ALAS-DNA; X59957 R. MELITOTI REC A ALAS-DNA     

Sensitivity variations of Listeria strains to the bacteriocins, lactocin 705, enterocin CRL35 and nisin

Five strains of Listeria monocytogenes, four strains of Listeria innocua and a strain of Listeria seeligeri showed different sensitivities to lactocin 705 (17 000 AU ml^–1), enterocin CRL35 (8500 AU ml^–1) and nisin (2500 IU ml^–1) at different pHs (5, 6 and 7). The susceptibility of Listeria strains to bacteriocins at each pH was strain dependent, and it was enhanced at the low pH. L. monocytogenes had enhanced nisin tolerance while the non-nisin bacteriocins were more inhibitory with viability losses of 3–3.4 in contrast with 1.5–1.8 log cycles, respectively. Lower viability loss values were obtained with L. innocua strains with all three bacteriocins while L. seeligeri was more sensitive to nisin than to lactocin 705 or enterocin CRL35.     

Growth and solute composition of the salt-tolerant kallar grass [Leptochloa fusca (L.) Kunth] as affected by nitrogen source

A study was conducted to elucidate the effect of N form, either NH₄ ⁺ or NO₃ ^–, on growth and solute composition of the salt-tolerant kallar grass [Leptochloa fusca (L.) Kunth] grown under 10 mM or 100 mM NaCl in hydroponics. Shoot biomass was not affected by N form, whereas NH₄ ⁺ compared to NO₃ ^– nutrition caused an almost 4-fold reduction in the root biomass at both salinity levels. Under NH₄ ⁺ nutrition, salinity had no effect on the biomass yield, whereas under NO₃ ^– nutrition, increasing salinity from 10 mM to 100 mM caused 23% and 36% reduction in the root and shoot biomass, respectively. The reduced root growth under NH₄ ⁺ nutrition was not attributable to impaired shoot to root C allocation since N form did not affect the overall root sugar concentration and the starch concentration was even higher under NH₄ ⁺ compared to NO₃ ^– nutrition. The low NH₄ ⁺ (2 mM) and generally higher amino-N concentrations in NH₄ ⁺- compared to NO₃ ^–-fed plants indicated that the grass was able to effectively detoxify NH₄ ⁺. Salinity had no effect on Ca²⁺ and Mg²⁺ levels, whereas their concentration in shoots was lower under NH₄ ⁺ compared to NO₃ ^– nutrition (over 66% reduction in Ca²⁺; over 20% reduction in Mg²⁺), but without showing deficiency symptoms. Ammonium compared to NO₃ ^– nutrition did not inhibit K⁺ uptake, and the K⁺-Na⁺ selectivity either remained unaffected or it was higher under NH₄ ⁺ than under NO₃ ^– nutrition. Results suggested that while NH₄ ⁺ versus NO₃ ^– nutrition substantially reduced root growth, and also strongly modified anion concentrations and to a minor extent concentrations of divalent cations in shoots, it did not influence salt tolerance of kallar grass.     

Three native Pseudomonas fluorescens strains tested under growth chamber and field conditions as biocontrol agents against damping-off in alfalfa

Alfalfa (Medicago sativa) is one of the most important crops used in Uruguay for livestock feeding. Seedling diseases, particularly damping-off, are a critical factor which limits its establishment. Three native Pseudomonas fluorescens strains, UP61.2, UP143.8 and UP148.2, previously isolated from Lotus corniculatus, were evaluated to determine their efficacy as biological control agents for alfalfa seedling diseases in the field. Their compatibility with the alfalfa-Sinorhizobium meliloti symbiosis was also assessed. In growth chamber conditions seed inoculation with Pseudomonas strains did not affect different parameters of alfalfa-rhizobium symbiosis as shown by nodulation rate and shoot dry weight of plants. The presence of the commercial inoculant strains of S. meliloti did not impair colonization by the P. fluorescens and vice versa. In field trials the dynamics of rhizobial rhizospheric populations were not affected by the presence of P. fluorescens. Each P. fluorescens strain successfully colonized alfalfa roots at adequate densities for biocontrol activity. Results showed that P. fluorescens strains provided a 10–13% increase in the number of established plants relative to the control, an intermediate result compared to the fungicide treatment (24%). The alfalfa above-ground biomass was increased by 13% and 15–18% in the presence of the fungicide and P. fluorescens strains, respectively. Therefore, results from this study demonstrated that the three P. fluorescens strains provided effective control against soil-borne pathogens and suggest a potential use in the development of a commercial inoculant to be applied for the control of legume seedling diseases.     

Plant regeneration from seedling explants of perennial Glycine species

More than 5000 cultures, from 30 accessions of six Glycine species, were established to assess the rôle of plant genotype in the response to an agar-solidified culture medium containing B5 salts and vitamins, 3% w/v sucrose, 1.1 mg 1^–1 BAP and 0.005 mg 1^–1 IBA, already known to induce shoot regeneration in callus of G. clandestina. Shoot initiation was obtained in a variety of explants from G. canescens, G. falcata, G. latrobeana and G. tomentella. With the exception of G. latrobeana, development of buds into shoots followed transfer to B5-based medium with 0.2 mg^–1 BAP and 0.005 mg 1^–1 IBA. Shoots readily produced roots in hormone-free half-strength B5 medium. In G. latrobeana, both extension and rooting occurred on this medium. Shoot regeneration was obtained in 12 of 30 accessions evaluated, but one accession of G. canescens, G1171, produced shoots and plantlets at a consistently higher frequency than other accessions, with plantlet recovery in more than 70% of the cultures. Bud formation in callus of G. canescens G1171 also occurred if BAP was replaced by 1.0 mg 1^–1 kinetin, 2i-p or zeatin, albeit at a lower frequency.