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 Abscisic Acid on the Growth Pattern of the Shoot Apical Meristem and on Flowering in Chenopodium rubrum L.

Abscisic acid (ABA) at 1 x 10^–4 M or 3 x 10^–4 Mwas applied to the apical buds of Chenopodium rubrum plantsexposed to different photoperiodic treatments and showing differentpatterns of floral differentiation. Stimulation of growth inwidth of the apical meristem of the shoot and/or inhibitionof growth in length was obtained under all photoperiodic treatments.This change of growth pattern was followed by different effectson flowering. In non-induced plants grown under continuous light ABA stimulatedpericlinal divisions in the peripheral zone and the initiationof leaves as well as the growth in width of bud primordia. Inplants induced by two short days reduced growth of the meristemcoincided with ABA application. Longitudinal growth of the meristemwas inhibited in this case and only a temporary stimulationof inflorescence formation took place. In plants induced ata very early stage, ABA exerted a strong inhibitory effect onflowering. A permanent and reproducible stimulatory effect onflowering was obtained in plants induced by three sub-criticalphotoperiodic cycles if ABA was applied to apices released fromapical dominance. In this case formation of lateral organs andinternodes was promoted by ABA and was followed by stimulatedinflorescence formation. Gibberellic acid (GA2) at 1x 10^–4M or 3 x 10^–4 M brought about a similar effect on floweringas ABA, although the primary growth effect was different, i.e.GA₂ stimulated longitudinal growth. The effects of ABA and GA₂ on floral differentiation have beencompared with earlier results obtained from auxin and kinetinapplications. These growth hormones are believed to regulateflowering by changing cellular growth within the shoot apex.Depending on the actual state of the meristem identical growthresponses may result in different patterns of organogenesisand even in opposite effects on flowering. Shoot apex, flowering, photoperiodic induction, abscisic acid, gibberellic acid, Chenopodium rubrum L.     

Auxin-gibberellin relationships in their effects on hypocotyl elongation of light-grown cucumber seedlings VI. Promotive and inhibitory effects of kinetin

The interaction of kinetin with IAA and GA₃ on the elongationof hypocotyl sections of Cucumis sativus L. cv. National Picklingwas studied. Kinetin in the concentration range of 10^–7M to 10^–4 M markedly inhibited IAA-induced elongation,while in a lower range from 10^–10 M to 10^–8 M, itsynergistically enhanced IAA-induced elongation. Kinetin alonein this range had no effect. A 5-to 15-min pulse treatment seemsenough to induce the maximum effect for both inhibition andpromotion. Since the magnitude of the maximum inhibition dependedon the concentration and not on the duration of treatment, thereaction in the cell caused by kinetin seemed to be completedwithin a short period. Washing of the sections with distilledwater after kinetin treatment (30 min) did not significantlyeliminate the kinetin effect. This probably indicates that thebinding of kinetin molecules to a supposed acceptor is not reversible.Interaction of kinetin with GA₃ in their pretreatment effectson IAA-induced elongation shows that in the inhibitory concentrationrange, the kinetin effect was partly overcome by GA₃, and thatin the promotive range, the magnitude of the enhancement wasdetermined by kinetin regardless of the presence of GA₃. Theeffect of kinetin seems to dominate over that of GA₃ indicatingthat the modes of their pretreatment effects differ from oneanother. (Received June 24, 1977; )     

In vitro Propagation of Red Cabbage (Brassica oleracea L. var. capitata)

By manipulation of various growth regulators and physical conditions,plants have been regenerated from excised roots, stem segments,cotyledons, leaves, and callus cultures of red cabbage (Brassicaoleracea var. capitata) grown under in vitro conditions. Shootbuds were induced on isolated root segments (1 cm long) culturedon Murashige and Skoog's medium and the frequency of bud formationwas greatly enhanced by the addition of kinetin (0.5 part 10^–6).Callus obtained from the seeds, cotyledons, and hypocotyl segmentscultured on a medium fortified with 2,4-D (1 part 10^–6),kinetin (0.1 part 10^–6), and coconut milk (10%, v/v) hasbeen repeatedly subcultured. The callus is slow growing, andon transference to a kinetin (2 parts 10^–6) and IAA (2parts 10^–6) medium underwent morphogenesis to give riseto plants. The significance of the propagation of red cabbageby in vitro culture is pointed out.     

Effects of Plant Growth Regulators on Grain-filling and Yield of Rice

Effects of three phytohormones (IAA₁, GA₃ and kinetin) on grain-fillingand the pattern of ³²P translocation from individual leavesto grains were studied at intervals of 7 days during the progressof reproductive development of rice (Oryza saliva L. cv. Jaya).The plants were sprayed with 100 µg ml^–1 aqueoussolutions of the hormones at 100 days, when the plants wereentering the reproductive stage. Kinetin produced a pronouncedeffect on grain-filling as well as on ³²P mobilization fromindividual leaf to grains and increased yield, possibly by increasingleaf longevity. GA₃ and IAA also increased the grain-fillingand ³²P mobilization significantly over control but the effectswere less marked than those of kinetin. Oryza sativa L., rice, grain yield, translocation, growth regulators, gibberellic acid, indol-3-yl acetic acid, kinetin     

Haploid Plantlet Formation from Female Gametophytes of Ephedra foliata Boiss. in vitro

Female gametophytes (at the archegonial stage) excised fromyoung ovules of Ephedra foliata Boiss, were cultured on a basalmedium (Murnshige and Skoog's combinations of major and minorsalts, Iron source, vitamins, myo-inositol along with 2 percent sucrose and 10 per cent coconut milk) under aseptic conditions.Growth and morphogenetic responses of the explants to auxinswere compared at different concentrations and a study of theirinteractions with cytokinins has also been made. At 2 mg 1^–1,2, 4-D induced profuse callusing which subsequently producedroots. NAA at 4 mg 1^–1 was optimal for callus growth androoting. Combinations of 2,4-D and kinetin were more effectivein inducing roots and shoot buds than those of 2,4-D and benzylamino-purine (BAP). Addition of BAP (0.05 mg ^1–1) to themedium containing optimal concentrations of NAA resulted information of a large number of roots. Kinetin induced only rootingin the presence of 4 mg 1^–1 NAA. A high concentrationof BAP (8 mg 1^–1), stimulated shoot bud formation. Forthe further development of shoot buds, neither auxin nor cytokininwas needed. Cytological observations revealed the presence ofhaploid number of chromosomes, i.e. seven. Ephedra foliata, tissue culture, callus, regeneration, 2,4-dichlorophenoxyacetic acid, naphthalene acetic acid, kinetin, benzyl amino-purine     

Effects of Indol-3yl acetic Acid on Floral Induction and Apical Differentiation in Chenopodium rubrum L.

Indol-3yl acetic acid (10^–4M) was applied to the plumulesof Chenopodium rubrum. Effects on the anatomical structure andthe growth pattern in the apical meristem, as well as DNA synthesisand nucleolus size were investigated. When auxin is applied before or during photoperiodic inductionit inhibits DNA synthesis and meristematic activity. The axillarymeristem (i.e. a group of cells in the axils of the leaf primordia)is most affected. A similar inhibition of the axillary meristemwas also observed in non-induced control plants grown in continuouslight. Auxin applied simultaneously with photoperiodic inductioncounteracts the reduction of apical dominance in the apex andthus inhibits the onset of floral differentiation. Auxin appliedfollowing induction inhibits the previously-formed buds andmakes possible a more complete development of the apical flower. The dual effect of IAA on flowering, inhibitory and stimulatory,manifests itself as a growth response at different stages ofthe changing shoot apex.     

Abscisic Acid and Apical Dominance in Phaseolus coccineus L. The Role of Tissue Age

Abscisic acid (ABA) applied to the decapitated second internodeof runner bean plants enhanced outgrowth of lateral buds onlywhen internode stumps were no longer elongating. Applied toelongating internodes of slightly younger plants, ABA causesinhibition of bud outgrowth. Together with 10^–4 M indol-3-ylacetic acid (IAA), ABA stimulated internode elongation and interactedadditively in the inhibition of bud outgrowth. A mixture of10^–5 M ABA and 10^–6 M gibberellic acid (GA₃ ) causedsimilar effects on internode growth as IAA + ABA, but was mutuallyantagonistic in effect on growth of the lateral buds. Abscisic acid, apical dominance, gibberellic acid, indol-3yl acetic acid, Phaseolus coccineus, bean     

Effects of Cytokinin and Several Inorganic Cations on the Polyamine Content of Lettuce Cotyledons

Kinetin (4.7 x 10^–5 M) and 6-benzyladenine (2.22 x 10^–5M) were found to increase ca. 2-fold the putrescine contentin cotyledons of lettuce (Lactuca sativa L.) seedlings grownfor 3 days under fluorescent light. On the other hand, severalinorganic ions (K⁺, Na⁺, Ga⁺⁺, Mg⁺⁺) at a concentration of 3x 10^–2 M reduced the putrescine content. The combinationof kinetin with one of several inorganic ions at the same levelmarkedly increased the spermine content, but the putrescinecontent decreased; calcium and magnesium ions were less effective.The physiological significance of these findings is discussed. (Received July 4, 1982; Accepted November 6, 1982)     

Correlative Inhibition of Lateral Bud Growth in Pisum sativum L. and Phaseolus vulgaris L.: Studies of the Role of Abscisic Acid

The possibility has been investigated that abscisic acid (ABA)might act as a correlative inhibitor of lateral bud growth inPisum sativum and Phaseolus vulgaris. Application of ABA insmall quantities (2µg) to axillary buds on decapitatedplants of P. sativum caused appreciable inhibition of theirgrowth, and induced a compensatory growth of the bud on an adjacentnode. Application of this same quantity of ABA to axillary budson decapitated plants of Phaseolus vulgaris was without effect,but a high concentration in lanolin (1 mg g^–1) did substantiallyreduce bud outgrowth. Endogenous ABA-like substances in Phaseolusvulgaris, detected by bioassay and electron capture g.l.c.,were present in similar concentrations in shoot tips, lateralbuds on intact plants and lateral buds on plants decapitated24 h earlier. The effects of applied ABA suggested that it might be involvedin the mechanism of correlative inhibition in Pisum sativum,but it was not possible to test this hypothesis by determiningendogenous ABA levels in axillary buds because of their smallsize. The evidence presented here suggests that ABA is not acorrelative inhibitor in Phaseolus vulgaris even though at highconcentration it can inhibit the growth of axillary buds.     

Differentiation of Shoot Buds in vitro in Tissue 1Sisymbrium irio L.

Shoot bud formation was induced in the stem callus of Sisymbriumirio L., a Cruciferous plant. The callus was established onMurashige and Skoog medium with IAA (1?0 mg l^–1) and kinetin(0?5 mg l^–1). The effect of three purines (kinetin, 6-benzylaminopurine,and 6-methylaminopurine) incorporated singly along with IAAin MS medium was investigated. It was found that kinetin orMAP (3–5 mg l^–1) along with IAA (0?5 mg l^–1)were the most effective in inducing shoot bud formation. Adeninesulphate (10 mg l^–1) with kinetin (1?0 mg l^–1) alsoinduced bud differentiation. The morphogenetic potential of the callus to differentiate shootbuds was seemingly lost in 2 year old callus cultures. However,on successively subculturing on a regeneration medium shootbuds differentiated and the number of buds formed improved onfurther subculture. Two types of meristematic outgrowths were recognized: (i) arisingfrom superficial cells and (ii) arising from deep-seated cellsin the vicinity of tracheidal elements. However, both typesformed meristematic nodules on the surface of which shoot budsdifferentiated. Some embryoids were also recognized arisingsuperficially.     

Shoot Axillary Bud Morphogenesis in Kiwifruit (Actinidia deliciosa)

The annual cycle of kiwifruit [Actinidia deliciosa(A. Chev.)C. F. Liang et A. R. Ferguson var.deliciosacv. Hayward] shootaxillary bud (first-order axillary bud, FOAB) morphogenesisis described. FOABs developed quickly with the majority of budscales and leaf primordia present approx. 125 d after budbreak(dab). Mature FOABs had, on average, 23.2 bud scales and leafprimordia. Most second-order axillary structures were also presentapprox. 125 dab. During the growing season, the second-orderstructures developed into second-order axillary buds (SOABs)or remained as simple, dome-shaped meristems (SDSMs). At maturity,nearly all FOABs had four SOABs and, on average, 12.4 SDSMs.Most SDSMs were fused to the subtending leaf primordia, butsome SDSMs developed so that they were ‘free’ fromthe subtending leaf primordia. Third-order axillary meristems(third-order SDSMs) were observed in the axils of most SOABs,and, on average, there were 20.6 per FOAB. Our observationson the development of second-order axillary structures are consistentwith evocation in kiwifruit occurring earlier than the generally-acceptedtime of late summer. Actinidia deliciosa; bud morphogenesis; development; flowering; evocation     

Regulation of Branching in Decussate Species with Unequal Lateral Buds

In the decussate plants Alternanthera philoxeroides and Hygrophilasp. the opposite axillary bud primordia are of unequal sizefrom the time of their inception; the larger or + buds lie alongone helix and the smaller or – buds along another (helicoidalsystem). In decapitated plants of Alternanthera both buds grewout, but unequally; if the node was vertically split growthof the two shoots was more equal, and if the + buds were excisedgrowth of the – shoots approximately equalled that ofcontrol + shoots. In decapitated shoots of Hygrophila grownin sterile culture only one bud, the + or larger one, grew outat each of the upper nodes. In excised cultured nodes, also,only the + bud grew out; but if the nodes were split longitudinallyboth buds grew out, initially rather unequally. These experimentssupport the view that the regulation of branching in these specieshas two components, apical dominance and the dominance of thelarger (+) bud over the smaller (–) bud at the same node.The restriction of growth potentiality imposed on the –bud is not permanent but can be modified. Further correlativeeffects on bud outgrowth include those of the subtending leavesand of buds at other nodes.     

Agrobacterium rhizogenes Mediated Transformation of the Forage Legumes Medicago sativa and Onobrychis viciifolia

Three cultivars of M. sativa and one cultivar of O. viciifoliawere evaluated for their response to inoculation with A. rhizogenesstrain A4T (containing pRiA4b). A cultivar-dependent responsewas observed in M. sativa with 94%, 25%, and 4% of infectedstem explants producing transformed roots in the cultivars Vertus,Regen-S, and Rangelander, respectively. In O. viciifolia cv.Hampshire Giant, an explant-dependent response was observedwith 78% and 50% of seedling cotyledon and hypocotyl explantsresponding, respectively. Leaf explants failed to produce transformedroots. Transformed roots showed plagiotropic and negativelygeotropic growth on hormone-free agar MS medium. Productionof transgenic shoots from O. viciifolia root cultures occurredspontaneously. Recovery of transgenic plants from M. salivacv. Rangelander was achieved by transfer of callus (inducedon UM medium containing 2·0mg dm^–3 2,4-D and 0·25mg dm^–3 kinetin) to MS medium containing 0·5 ingdm^–3 BAP and 0·05 mg dm^–3 NAA. Cultured rootsof both species synthesized opines (agropine and mannopine).Extensive morphological variation was observed in plants ofM. sativa (clone Al) and O. viciifolia (clone A4Tl) establishedin the glasshouse. DNA sequences homologous to TL-DNA and TR-DNAwere present in root clones and regenerated plants. Key words: Agrobacterium rhizogenes, Medicago sativa, Onobrychis viciifolia, transformed roots, transgenic plants     

Organogenesis in the Cultured Female Gametophyte of Ephedra foliata

The female gametophyte of Ephedra foliata was used as an explantfor the production of haploids as it is composed of haploidcells, all of the same genotype. The regeneration of roots wasdependent upon the presence of NAA, while BAP had a modifyingeffect. At lower concentrations (0.05 parts 10^–6 and 3.5parts 10^–6) BAP enhanced the root promotion of NAA (0.05–4.0parts 10^–6). At higher concentrations of BAP (1–6parts 10^–6), roots and shoot buds were formed. Kinetinat 4.0 parts 10–6 with 0.5 parts 10–6 2, 4-D wasoptimal for shoot bud production in explants at the archegonialstage and 2, 4-D at 2.0 parts 10–6 with 0.5 parts 10–6kinetin was optimal for root formation. Cells of the callusand root tip had the haploid number of chromosomes, n = 7. Meristemoidswere located on the surface or embedded in the callus tissue.The deep seated meristemoids organized only root primordia,but the peripheral ones gave rise to root as well as shoot budprimordia. Initially, there was no vascular connection betweenthe shoot-bud and the callus. This was established later. Key words: Ephedra, Female gametophyte, Haploid, Tissue culture     

In Vitro Morphogenesis in Nardostachys jatamansi DC.: Shoot Regeneration from Callus Derived Roots

Callus cultures of Nardostachys jatamansi DC. maintained onMurashige and Skoog's medium containing 3.0 mg 1^–1 of-naphthaleneacetic acid and 0.25 mg 1^–1 of kinetin whenshifted to medium containing 0.25–1.0 mg 1^–1 ofindole-3-acetic acid or indole-3-butync acid showed profuserhizogenesis. The callus-regenerated roots when transferredto medium containing 2.0–6.0 mg 1^–1 of kinetin producedshoot buds. The de novo shoot bud regeneration took place eitherdirectly from cortical cells or from the inner stelar region.In addition, direct, concomitant root-shoot development wasalso observed. Nardostachys jatamansi, organogenesis, root-buds     

Involvement of cellulose synthesis in actions of gibberellin and kinetin on cell expansion. Gibberellin-coumarin and kinetin-coumarin interactions on stem elongation

In azuki bean (Azukia angularis = Vignia angularis) epicotylsections, 5 ? 10^–4 M coumarin inhibited the incorporationof radioactivity from [U^–14C]glucose into the cellulosefraction by 35% in the absence of indole-3-acetic acid (IAA)and by 40% in the presence of 1 ? 10^–4 M IAA. There wasno inhibitory effect on the incorporation of radioactivity intothe other fractions. Coumarin at 5 ? 10^–4 M reversed thepromoting effect of 1 ? 10^–5 M gibberellin A₃ (GA) andthe inhibitory effect of 1 ? 10^–5 M kinetin on IAA-inducedelongation of sections with no significant effects on IAA-inducedelongation. Neither GA nor kinetin had any appreciable effectson cellulose synthesis. No inhibition of cellulose syntheiswas observed with 1 ? 10^–3 M colchichine, which has beenreported to have effects similar to those of coumarin on GA-or kinetin-affected stem elongation. Coumarin at 5 ? 10^–4M was ineffectual in breaking up wall microtubules, while adisrupting effect on wall microtubules was clearly demonstratedwith 3 ? 10^–4M colchicine. From these results, the possible involvement of cellulose synthesisin cell expansion controlled by GA or kinetin was suggested. (Received August 3, 1973; )     

Kinetin Transport to Axillary Buds of Dwarf Pea (Pisum sativum L.)

Decapitation resulted in the transport of significant amountsof ¹⁴C to the axillary buds from either point of application,but pretreatment of the cut internode surface of decapitatedplants with IAA (alone or in combination with unlabelled kinetin)inhibited the transport of label to the axillary buds and resultedin its accumulation in the IAA-treated region of the stem. Inintact plants to which labelled kinetin was applied to the apicalbud there was little movement of ¹⁴C beyond the internode subtendingthis bud; when labelled kinetin was applied to the roots ofintact plants, ¹⁴C accumulated in the stem and apical bud butwas not transported to the axillary buds. A considerable proportionof the applied radioactivity became incorporated into ethanol-insoluble/NaOH-solublecompounds in the apical bud of intact plants, in internodestreated with IAA, and in axillary buds released from dominanceby removal of the apical bud. The results are discussed in relation to the possible role ofhormone-directed transport of cytokinins m the regulation ofaxillary bud growth.     

Effect of Photoperiodic Induction on the Transpiration Rate and Stomatal Behaviour of Debudded Xanthium Plants

In controlled-environment studies with debudded Xanthium plants,appreciable changes in stomatal activity and attendant ratesof transpiration were found to be associated with photoperiodicinduction. Leaves of plants kept on a 10-h inductive photoperiodafter removal of the apical and axillary buds grew at ratescomparable to those of similarly debudded plants kept on a 10-h-interruptednon-inductive photoperiod (consisting of an 8-h photoperiodand a 2-h interruption of the dark period). There was a large effect of leaf age on stomatal behaviour:the minimum stomatal resistance of leaves of non-induced plantsdecreased from about 5 s cm^–1 when the leaf was 25 percent of its final size to 1.6 s cm^–1 when almost fullyexpanded. Thereafter, it slowly increased with time. Superimposedon this age response was a marked effect of photoperiodic induction.The stomata on induced leaves opened more widely than thoseon non-induced leaves, the response being greatest with theleaves which were youngest at the time induction commenced.However, this ‘opening’ tendency was maintainedfor only a few weeks; thereafter, the stomata failed to openas widely. This later ‘closing’ tendency of stomataon leaves of induced plants progressed rapidly and in a basipetalsequence and presaged a necrotic form of leaf senescence whichdeveloped in the same sequence. The closing tendency on leavesof non-induced plants progressed slowly in an acropetal direction;leaves senesced in the same sequence with the familiar yellowingsymptoms. It is suggested that flower induction sets in train a sequenceof events which influence stomatal movement (and other processes)and inevitably leads to the death of the induced axis. Transpiration rates calculated from measurements of the physicalenvironments and stomatal resistances agreed well with thosemeasured.     

Effect of External Supply of Growth Substances on Axillary Proliferation and Development in Dioscorea bulbifera

Bulbil development in cultured nodes of D. bulbifera proceededin the absence of growth substances from the medium. When IAAwas incorporated into the medium at the concentrations of 5mg l^–1 and 10 mg l^–1 the cultured nodes producedlarger bulbils than in its absences. When the concentrationof IAA was increased to 15 mg l^–1, however, the culturednodes produced a callus instead of a properly organized bulbil.The dry weight of bulbils increased when kinetin was added tothe medium at the concentrations of 0.05, 0.5, and 2.5 mg l^–1.The greatest increase was with 0.5 mg l^–1 kinetin. Onincreasing the concentration of kinetin in the medium to 5.0mg l^–1 the tissue produced had smaller dry weight thanthose produced in the absence of growth substances. Additionof different combinations of IAA and kinetin to the basal mediumresulted in the production of normal bulbils, roots, and shootsin some instances (suitable combinations) and in the productionof callus and abnormal shoots in others (non suitable combinations).