Symbiotic effectiveness of Bradyrhizobium strains isolated from Parasponia and tropical legumes on Parasponia host species

The symbiotic effectiveness of Bradyrhizobium strains isolated from three species of Parasponia and from legumes were compared on Parasponia grown in Leonard-jars. Effectiveness of each symbiotic association was estimated from dry weight and total nitrogen of shoots and nodules of plants grown on medium free of combined nitrogen. Twenty strains isolated from three species of Parasponia were found to vary in their effectiveness on P. andersonii, the least effective fixing one fifth of the nitrogen of the most effective strains. The outcome of the symbiosis was not associated with the host source of the test strain. P. andersonii, P. rugosa and P. rigida responded differently to a selection of seven strains of Parasponia Bradyrhizobium; some strains were either ineffective or fully effective on each host, while others varied in their symbiotic performance. P. andersonii fixed significantly (P < 0.001) larger quantities of nitrogen than either P. rugosa or P. rigida with p. rigida being the least effective. In contrast to Bradyrhizobium strains from Parasponia spp. which formed nodules rapidly (within 11–20 days), nine strains isolated from legumes required between 25 and 74 days to form partially effective nodules. The thre Parasponia species formed relatively large quantities of nodule tissue relative to the amount of nitrogen fixed and shoot dry matter produced. The Bradyrhizobium isolated from Parasponia plants growing in Papua New Guinea soils could be grouped together on the basis of their infection characteristics on Parasponia and legumes.     

Polyamines in Nodules from Various Plant-Microbe Symbiotic Associations

Polyamine compositions of root or stem nodules collected fromvarieties of nitrogen-fixing leguminous (22 species) and non-leguminous(5 species) plants were investigated. Relatively high concentrationsof homospermidine were observed in root or stem nodules of allthe leguminous plants. Based on the ratio of homospermidineto spermidine, legume nodules were generally characterized intotwo major groupes; one containing almost equal amounts of homospermidineand spermidine, and the other a high homospermidine/spermidineratio. Root nodules from pigeon pea (Cajanus cajan L. Millsp)was the only exception which exhibited very low homospermidine/spermidineratio. Amongst the legumes, nodules of adzuki bean (Vigna angularis),siratro (Macroptilium atropurpureum DC. Urb.), pea (Pisum sativumL.), and hairly vetch (Vicia hirsuta S.F. Gray) were rich indiamine putrescine. Such characters of nodule polyamine compositionwere inherent characteristics of each legume species, and notrelated to the type of infected rhizobia (Rhizobium or Bradyrhizobium).In contrast to herbaceous leguminous plants, nonleguminous woodyplants, which symbiotically associate with actinomycete Frankiaspecies, contained little polyamines in their root nodules.Root nodules of non-leguminous Parasponia andersonii infectedby bradyrhizobia were found to contain large quantities of putrescineand homospermidine. No significant differences in polyaminecomposition were observed between root and stem nodules bothin Aeschynomene indica and Sesbania rostrata. (Received June 13, 1994; Accepted August 17, 1994)     

The nodulation of micro-propagated plants of Parasponia andersonii by tropical legume rhizobia

A simple clonal micro-propagation system for Parasponia andersoniiwas employed to study the nodulation response of this non-legumeto inoculation by the broad host range Rhizobium sp. NGR234,isolated from Lablab purpureus, and also to tropical legumerhizobia isolated from Aeschynomene species. Partially effectivenodules, assayed by acetylene reduction and ¹⁵N dilution procedures,were induced with strain NGR234 and its spontaneous streptomycinresistantmutant ANU240. Effective nodules were produced by one of theAeschynomene strains (ORS302) tested, with rates of acetylenereduction comparable to those of root nodules produced by Bradyrhizobiumstrain CP279, originally isolated from P. andersonii. Lightand transmission electron microscopy showed that there was acorrelation between the nitrogen fixing capability of the symbiosisbetween NGR234 and Parasponia and the number of persistent infection(fixation) threads within the nodule cells. Key words: Parasponia, Bradyrhizobium, Rhizobium, Aeschynomene, micro-propagation, root nodules, nitrogen fixation     

In Vitro Multiple Shoot Regeneration in Syzygium aromaticum

Multiple shoots were induced from nodal segments of seedlingsof Syzygium aromaticum, on Murashige and Skoog's (MS) basalmedium at half strength salts and Gamborg's medium (B5), supplementedwith BAP and NAA. Six to eight shoots were obtained when 3 mgl^–1 BAP and 0.5 mg l^–1 NAA were used in the medium.Both MS medium and B5 medium showed more or less similar resultsregarding the proliferation of the explants. Syzygium aromaticum (L) Merr and Perry (clove), multiple shoots, regeneration     

Direct Organogenesis and Plant Regeneration in Preconditioned Tissue Cultures of Lathyrus cicera L., L. ochrus (L.)DC. and L. sativus L.

This study demonstrates the importance of preconditioning ofsource tissue in regeneration of multiple shoot buds from severalspecies of Lathyrus. Preconditioned multiple shoots of Lathyruscicera L., L. ochrus (L.) DC. and L. sativus L. were obtainedby germinating seeds on Murashige and Skoog (MS) medium containing50 µM N⁵-benzylaminopurine (BAP) for 2 to 3 weeks. Multipleshoot bud formation occurred when epicotyl explants of preconditionedshoots were cultured on MS medium containing 5–50 µMBAP. No shoot regeneration was observed from epicotyl explantswhich were obtained from non-preconditioned shoots. Shoot budswere formed directly on explants without an intervening callusphase after 2 to 3 weeks of culture. Regenerated shoot budsformed healthy shoots which developed prolific and strong rootswhen transferred to MS medium supplemented with 2.5 µMnaphthaleneacetic acid (NAA). Lathyrus cicera L., L. ochrus (L.) DC., Ochrus Vetch, L. sativus L., Lathyrus pea, de novo differentiation, epicotyl, preconditioning with BAP     

TheParasponia parviflora — Rhizobium symbiosis. Isotopic nitrogen fixation,hydrogen evolution and nitrogen-fixation efficiency,and oxygen relations

Summary Isotopic¹⁵N₂ experiments confirmed nitrogen fixation inParasponia parviflora. The conversion ratio C₂H₄/N₂ was 6.7 under the experimental conditions employed. Measurements of the δ¹⁵N in leaves of Parasponia and Trema showed on the basis of these determinations thatParasponia parviflora possesses N₂-fixing capacity and can be distinguished in this respect from the non-nitrogen-fixingTrema cannabina tested by the same method. Therefore, δ¹⁵N can be used to monitor N₂ fixation in natural ecosystems. Hydrogen evolution and the relative efficiency of N₂ fixation in this relation have been determined. DetachedParasponia parviflora root nodules grown in soil and tested in an argon/oxygen atmosphere produced appr. 4 μmol H₂.h⁻¹.g⁻¹ fresh weight root nodules. The relative efficiency of hydrogen utilization as measured in argon, air, and in the presence of C₂H₂ 10% (v/v) was for both equations used for to express this efficiency 0.96 and 0.97, respectively. This indicates that Parasponia like the root nodules of some actinorhizal symbioses (Alnus, Myrica, Elaeagnus) and some tropical legumes (Vigna sinensis) has evolved mechanisms of minimizing net hydrogen production in air, thus increasing the efficiency of electron transfer to nitrogen. The oxygen relation of nitrogen fixation (C₂H₂) inParasponia parviflora root nodules was determined. The nitrogenase activity of Parasponia root nodules increased at increasing oxygen concentrations up till c. 40% O₂. At oxygen levels above 40% O₂, the nitrogenase activity of the root nodules was nil or very erratic suggesting that at these oxygen levels the nitrogenase is not longer protected against the harmful effect of oxygen. In this respect Parasponia root nodules differ from actinorhizal root nodules in other nonlegumes, where optimal nitrogenase activity was observed in the range of 12–25% oxygen. Respiration experiments with Parasponia root nodules showed that in the range 10, 20, and 40% oxygen, the respiration rate (CO₂ evolution) increased concomitantly with an increase of the acetylene reduction rate. The CO₂/C₂H₄ values obtained varied between 8.1 and 19.2, being therefore 2–3 times higher than similar estimations in some actinorhizal and legume root nodules. The respiratory quotient (RQ) of detachedParasponia parviflora root nodules was in air initially approximately 2.0, but this value dropped to about 1.0 in a 3-hours period.     

Efficiency of Agrobacterium rhizogenes–mediated root transformation of Parasponia and Trema is temperature dependent

Parasponia trees are the only non-legume species that form nitrogen-fixing root nodules with rhizobium. Based on its taxonomic position in relation to legumes (Fabaceae), it is most likely that both lineages have gained this symbiotic capacity independently. Therefore, Parasponia forms a bridging species to understand the evolutionary constraints underlying this symbiosis. However, absence of key technologies to genetically modify Parasponia seriously impeded studies on these species. We employed Agrobacterium rhizogenes to create composite Parasponia andersonii plants that harbour transgenic roots. Here, we provide an optimized protocol to infect P. andersonii as well as its non-symbiotic sister species Trema tomentosa with A. rhizogenes. We show that the transformation efficiency is temperature dependent. Whereas the optimal growth temperature for both these species is 28?°C, the transformation is most efficient when co-cultivation with A. rhizogenes occurs at 21?°C. Using this optimized protocol up to 80?% transformation efficiency can be obtained. These robust transformation platforms will provide a strong tool to unravel the Parasponia–rhizobium symbiosis.     

Root-nodule symbiosis between Rhizobium and Parasponia (Ulmaceae)

Summary Evidence of root nodulation and N₂ fixation byParasponia parviflora Miq. found earlier by Akkermanset al. ^{1, 2} was confirmed, although with a lower value for N₂ fixation. In contrast to the above-mentioned investigation, theParasponia trees examined in the present study were growing under natural conditions.The evidence giving by Akkermanset al. ^{1, 2}, that Trinick's Trema is aParasponia species was confirmed by cytological and structural studies of Parasponia root nodules, which resembled closely the Trema root nodules of Trinick¹⁹. Moreover, evidence was obtained thatTrema cannabina Lour. growing in natural habitat on Java and two imported, hitherto unexamined,Trema species (T. micrantha andT. guineensis) bore no root nodules.Isolates fromParasponia parviflora root nodules produced effective root nodules both in sterile Parasponia seedlings and sterileVigna unguiculata (cowpea) andMacroptilium atropurpureum plants confirming their identity as Rhizobium.     

Micropropagation of Indian Rosewood by Tissue Culture

Multiple shoots were induced on excised hypocotyl segments andshoot tips of in vitro germinated seedlings of Indian rosewood(Dalbergia latifolia) on Murashige and Skoog's medium supplementedwith cytokinins and auxins. Roots were induced when individualshoots were treated first with half strength MS medium supplementedwith NAA, IAA and IBA (1 mg 1^–1 each) and subsequentlytransferred to hormone-free half-strength MS medium. The plantletswere then transferred to pots and grown in the greenhouse. Dalbergia latifolia, micropropagation, tissue culture, clonal propagation     

Translocation of iron to the N2-fixing stem nodules of Sesbania rostrata (Brem)

Labelled iron (applied as ⁵⁵FeCl₃) moves rapidly from the soilto the shoots and stem nodules of S. rostrata. Time-course experimentsare described that investigate the movement of iron throughdifferent plant parts, detailing the route of iron translocation.The results show that iron moves first to the mature leavesand is subsequently translocated to the vegetative buds andstem nodules. Phloem girdling the stem nodules almost completelystops the movement of labelled iron into the nodules. We concludethat iron is supplied to stem nodules in the phloem; these resultssupport other work suggesting there is no inward xylem flowinto legume nodules, which probably receive almost all nutrientsand most of their water via phloemmediated transport. Key words: Translocation, nutrient, Fe, iron, stem nodules, legume, nitrogen fixation     

Shoot and Plantlet Formation in Organ and Callus Cultures of Albizzia lebbek Benth

Plantlets were produced in vitro from root and hypocotyl explantstaken from seedlings of the tree legume, Albizzia lebbek. Theseexplants formed shoots when cultured with 5.0 mg l^–1 kinetinand 1.0 mg l^–1 IAA in MS medium. Shoots were also inducedin large numbers from callus treated with benzylaminopurine.About 20 per cent of the shoots rooted and were grown into plants. Albizzia lebbek Benth, tree legume, hypocotyl, root, in vitro cultures, shoot-plantlet induction     

In Vitro Culture and Plant Regeneration in Vicia faba subsp. Equina (var. Spring Blaze)

Explants of stem, leaves, roots, and cotyledons from etiolatedaxenically grown Vicia faba seedlings were cultured on a rangeof media. Shoot organogenesis was only obtained with nodal stemand cotyledonary node explants when cultured on MS medium with3% sucrose, 2.0 mg 1^–1 BAP and 02 mg 1^–1 NAA. Callusproliferation accompanied shoot organogenesis from nodal stemexplants. Successive subculture of nodal stem callus resultedin proliferation of regenerative callus which contained severalshoot bud initials. The capacity for shoot regeneration fromthis callus was maintained for 9 months. Histological studiesreveal de novo formation of meristematic centres in callus andtheir further development into bud primordia. High frequencyrooting of these adventitious shoots was obtained on half-strengthMS medium with 1.5% sucrose, 0.1 mg 1^–1 NAA and 0.5 mg1^–1 kinetin. Key words: Vicia faba, adventitious shoots, axillary shoots, de novomeristem formation, organogenesis, tissue culture     

Effects of Sucrose Concentration on the Photosynthetic Ability of Rose Shoots In Vitro

Reducing the concentration of sucrose in the culture mediumover successive subcultures has been tested as a method forincreasing the ability of rose shoots grown in vitro (Rosa cvsIceberg and Peace) to take up CO₂. Shoots maintained on ‘constant’10, 20 and 40 g I^–1 sucrose showed decreased levels ofCO² uptake at higher sucrose concentrations, although cv. Peacegrew least at 10 g l^–1 and showed correspondingly lowamounts of CO₂ uptake compared with 20 and 40 g l^–1. Bothcultivars died when sucrose was omitted from the medium. Assucrose concentration was reduced in the medium, so CO₂ uptakeof shoots initially cultured on 20 and 40 g l^–1 sucrosewas found to increase, although a concentration of 10 gl ^–1sucrose seemed to be limiting, below which the growth and chlorophylllevels of shoots declined. Rosa hybrid, rose, shoot culture in vitro, photosynthetic ability, sucrose, infra-red gas analysis     

Cyclic AMP in Rhizobia and Symbiotic Nodules

Cyclic adenosine 3',5'-monophosphate (cAMP) content of variouscultured rhizobia strains and tissues of legumes and non-leguminousplants was measured by enzyme immunoassays. Most rhizobia, culturedfor 44 to 165 h, contained cAMP ranging from 0.6 to 5 pmol mg^-1proteinexcept forAzorhizobium caulinodansORS571. The culture mediaalso contained varying amounts of cAMP depending on the strainof rhizobia.Azorhizobiumcells and their media contained no detectablecAMP. Nodules from most legumes and non-legumes had cAMP contentsranging from 2–70 pmol g^-1f.wt. However, nodules fromSesbaniarostrata,Crotalaria spectabilisandParasponia andersoniishowedundetectable cAMP levels, and those fromGlycine maxandVignaangularisoccasionally showed levels below the detection limit.The leaves of non-legumes mostly had cAMP levels below detectionlimit (approx. 1.0 pmol g^-1 f.wt), while the leaves ofa few legumes occasionally had detectable cAMP. The possiblerole of cAMP as a symbiotic signal is discussed. cAMP; legumes; modules; rhizobia; symbiosis     

Production of Plantlets of Shorea roxburghii G. Don. from Embryonic Axes Cultured In Vitro

Development of axillary shoots was induced in embryonic axesof the dipterocarp Shorea roxburghii G. Don. cultured on a modifiedMS medium containing 6-benzyl-aminopurine (BAP) at an optimumconcentration of 5 mg I^–1. Excised axillary shoots wereused in multiplication and rooting experiments. Vigorous rootdevelopment occurred in shoots supported on filter paper bridgesin liquid medium containing naphthaleneacetic acid (NAA) andindolebutyric acid (IBA) (0.1 mg I^–1 each). Shorea roxburghii, Dipterocarpaceae, tissue culture, plantlet formation     

Growth of Carnation Meristems in vitro: Anatomical Structure of Abnormal Plantlets and the Effect of Agar Concentration in the Medium on their Formation

Carnation meristems cultured in vitro grow into shoots of threetypes: normal, translucent and succulent. The apical meristemof succulent shoots was of the mantle-core type and it lackedpro-vascular tissue. The leaf had large vacuolated mesophyllcells, fewer stomata (often plugged), and no plate meristem.A higher agar concentration in the medium increased the percentageof normal shoots developing. This supports other indicationsthat the water potential of the medium affects morphogenesis. carnation, Dianthus caryophyllus L, meristem culture, abnormal plantlets, shoot meristem     

The Cobalt Requirement of Non-legume Root Nodule Plants

As already shown for Alnus glutinosa, cobalt is found to beessential for the proper growth of nodulated plants of Casuarinacunninghamiana and Myrica gale in a nitrogen-free rooting medium.If cobalt is not supplied, the plants develop symptoms of nitrogendeficiency; under the conditions of the experiments such symptomsbecame pronounced during the second season of growth of theseperennial plants. No cobalt requirement could be detected innon-nodulated plants of Alnus and Myrica supplied with nitrateor ammonium-nitrogen, and this suggests that in nodulated plantsthe need for cobalt is confined to the nodules. Vitamin B₁₂analogues are shown to be present in the nodules in relativelylarge amounts when cobalt is supplied, their formation beingattributed to the endophytes, which may therefore require cobaltfor their growth. The great reduction in fixation of atmosphericnitrogen in cobalt-deficient nodules may be due to a retardedgrowth of the endophyte, though this is not the only possibility.The cobalt relation of these non-legumes appears to be basicallysimilar to that of legumes.     

In vitro Shoot and Root Formation in the Apple Cultivar Akero

Regulation of in vitro shoot and root formation and the histologicalorigin of newly formed shoots was studied in the apple cultivarÅkerö. Both composition of mineral elements and benzylaminopurine(BAP) concentration affected shoot multiplication. Similar numbersof shoots were obtained with Lepoivre and MS medium after twosucceeding subcultures, but Murashige and Skoog medium was preferabledue to production of longer shoots. The optimum BAP concentrationwax around 8.8 µM. Higher concentrations caused vitrifiedshoots. The rooting ability increased with numbers of subcultures.Also the concentration of indol-3-yl butyric acid (IBA) affectedrooting. A strong interaction between numbers of subculturesand IBA concentration was obtained. After insufficient numbersof subcultures, when shoots were still difficult to root, increasingIBA concentration exerted little effect on rooting. When shootshad reached an ‘easy-to-root condition’ root initiationdepended on IBA concentration, showing an optimum at 2.5 µM.Supraoptimal IBA concentrations delayed root initiation. Dark treatment of shoots during the root-initiation phase increasedrooting ability. The most positive effect was obtained at suboptimalIBA concentrations. Anatomical studies revealed both axillaryand adventitious shoots. Two kinds of adventitious structureswere demonstrated. Malus domestica Borkh. apple cultivar Åkerö, in vitro propagation, anatomy, origin of shoots     

Studies on the Physiology of Nodule Formation: With two Figures in the Text: A Reappraisal of the Effect of Preplanting

Nutman (Arm. Bot. 21, 321, 1957) found that preplanting agarslopes with Trifolium pratense L. or Medicago sativa L. advancedthe time when second lots of plants of these species grown onthe same slopes initiated nodules, and depressed the total numberof nodules formed. He attributed these effects to root secretionswhich at low concentration hasten initial nodulation but athigher concentrations inhibit nodule formation. Further workhas now shown that initial nodulation is stimulated becausethe preplant removes traces of nitrate from the medium. Theamount of nitrate in the tap water used to prepare the medium(6?5 p.p.m. N) also increases the number of nodules formed onthe control plants, and this effect explains to a considerableextent the depression of nodule numbers by preplanting. Initial nodulation was delayed by small amounts of nitrate andnitrite but not by other forms of combined nitrogen (ammonium,asparagine, and urea). All forms of combined nitrogen testedincreased the number of nodules formed over a period of 8 weekswhen supplied at an initial concentration of 20 p.p.m. N.     

In vitro Regeneration of Mustard Plants (Brassica juncea var. RAI-5) on Cotyledon Explants from Non-irradiated, Irradiated and Mutagen-treated seed

Shoot formation in cotyledon explants of mustard (Brassica junceavar. Rai-5) was observed on Murashige and Skoog's medium supplementedwith NAA^* (1 mg l–1) and BA (1 mg l–1). Hypocotylsegments failed to differentiate shoots. Complete plants wereobtained when shoots were rooted in MS medium with NAA (1 mgl–1). EMS, a chemical mutagen, had an inhibitory effecton shoot regeneration. Gamma rays in doses above 2 kR suppressedshoot regeneration but stimulated callus growth. Brassica juncea, mustard, regeneration, tissue culture