Hormonal Regulation of Lateral Bud (Tiller) Release in Oats (Avena sativa L.)

Stem segments containing a single node and quiescent lateral bud (tiller) were excised from the bases of oat shoots (cv. `Victory') and used to study the effects of plant hormones on release of lateral buds and development of adventitious root primordia. Kinetin (10⁻⁵ and 10⁻⁶ molar) stimulates development of tillers and inhibits development of root primordia, whereas indoleacetic acid (IAA) (10⁻⁵ and 10⁻⁶ molar) causes the reverse effects. Abscisic acid strongly inhibits kinetin-induced tiller bud release and elon-gation and IAA-induced adventitious root development. IAA, in combination with kinetin, also inhibits kinetin-induced bud prophyll (outermost leaf of the axillary bud) elongation. The IAA oxidase cofactor p-coumaric acid stimulates lateral bud release; the auxin transport inhibitor 2,3,5-triiodo-benzoic acid and the antiauxin α (p-chlorophenoxy)-isobutyric acid inhibit IAA-induced adventitious root formation. Gibberellic acid is synergistic with kinetin in the elongation of the bud prophyll. In intact oat plants, tiller release is induced by shoot decapitation, geostimulation, or the emergence of the inflorescence. Results shown support the apical dominance theory, namely, that the cytokinin to auxin ratio plays a decisive role in determining whether tillers are released or adventitious roots develop. They also indicate that abscisic acid and possibly gibberellin may act as modulator hormones in this system.     

IN VITRO CULTURE OF FLORAL BUDS OF AQUILEGIA

Tepfer, S. S., R. I. Greyson, W. R. Craig, and J. L. Hindman. (U. Oregon, Eugene.) In vitro culture of floral buds of Aquilegia. Amer. Jour. Bot. 50(10): 1035–1045. Illus. 1963.—Floral buds at various stages of development, from early stages before sepal initiation to late stages with young carpel primordia present, were grown in culture on various agar media. A basic medium containing White's minerals, Nitsch's trace elements, coconut milk, sucrose, and assorted water-soluble vitamins was developed for growth of the buds. The addition of indoleacetic acid, gibberellic acid, and kinetin to the basic medium extended the developmental limits of buds at nearly all stages and decidedly improved the continued development of carpels. On this medium buds produce all of their organ primordia, growing from early stages to about the size of flowers at anthesis, but will not develop unless the sepals are removed. This inhibiting effect of sepals is not understood at this time. Stamen development is consistently poor beyond the point of differentiation of anther and filament, even with the addition of hormones. The development of buds in culture is illustrated and compared with development in intact plants. With further improvement of the medium, it is hoped that these buds may be used for experiments testing theories of floral morphogenesis.     

Tissue culture and plant regeneration of okra (Abelmoshus esculentus)

Explants (hypocotyl, cotyledon, cotyledonary node and leaf segment) were excised from aseptically grown okra (Abelmoschus esculentus) seedlings. The explants were cultured on a Murashige and Skoog basal nutrient medium supplemented with auxins, cytokinins and auxin-cytokinin combinations. Callus formation and root differentiation occurred in a medium containing naphthaleneacetic acid (NAA) or indoleacetic acid. There was a greater proliferation of roots on medium supplemented with NAA. The addition of 2,4-dichlorophenoxyacetic acid (2,4-D) to the growth medium suppressed root formation. No shoot bud or shoot development was observed at any of the auxin levels tested. Both kinetin (KN) and zeatin (Z) also proved ineffective in inducing shoot buds or shoots. Shoots were produced on cotyledon and cotyledonary node explants cultured in a medium supplemented with benzyladenine and NAA. These shoots developed roots on the same medium. The plantlets, on transfer to soil, grew normally.     

CHEMICAL CONTROL OF ADVENTITIOUS ORGAN FORMATION IN LACTUCA SATIVA EXPLANTS

Both kinetin and adenine promote bud initiation in excised cotyledons of Lactuca sativa. Controls lacking these substances form abundant roots but have never formed buds. Indoleacetic acid and certain mineral salts are also necessary for regeneration of shoots. Although bud growth from cotyledons is extensive on a medium containing both indoleacetic acid and kinetin, excised roots display a low propensity toward bud formation and typically develop callus tissue with roots. Growth of hypocotyl sections is intermediate with respect to bud formation. Shoot initiation in lettuce thus varies with the region of the seedling as well as with the culture medium.     

EFFECTS OF GROWTH SUBSTANCES ON EXCISED FLORAL BUDS OF AQUILEGIA

Buds at various stages of development were grown for 3 weeks on solid media containing coconut milk, minerals, vitamins, sucrose, and varying quantities of gibberellic acid, indoleacetic acid, and kinetin. The best average growth was obtained on media containing GA at 2.0 mg/liter, IAA at 1.0 mg/liter, and kinetin at 0.05 mg/liter. When results were compared for buds explanted at very young stages and at older stages, however, young buds attained better average growth on media with 0.5 mg/liter of IAA. Evidence is presented for interaction between IAA and kinetin. With buds explanted at young stages, as the kinetin concentration was increased, optimum growth occurred on media with increasing concentrations of IAA. With older buds, on the other hand, as the kinetin concentration was increased, optimum growth occurred on decreasing concentrations of IAA. Bud growth was compared on media with growth substances sterilized by autoclaving with growth on similar media with these substances filter-sterilized. Better growth occurred generally on the media with filter-sterilized ingredients. A long-range objective of this research is the development of a system that would make possible quantitative measurements of floral development in vitro.     

Effect of 2,4-Dinitrophenol on Auxin-induced Ethylene Production and Auxin Conjugation by Mung Bean Tissue

Auxin-induced ethylene production by mung bean (Phaseolus mungo L.) hypocotyl segments was markedly inhibited by 2,4-dinitrophenol regardless of whether or not kinetin was present. Uptake of indoleacetic acid-2-¹⁴C was also inhibited in the presence of 2,4-dinitrophenol. Segments treated only with indoleacetic acid rapidly converted indoleacetic acid into indole-3-acetylaspartic acid with time whereas kinetin suppressed indoleacetic acid conjugation. Formation of indole-3-acetylaspartic acid was significantly reduced when 2,4-dinitrophenol was present. The suppression of indoleacetic acid conjugation by kinetin and 2,4-dinitrophenol appeared to be additive, and the free indoleacetic acid level in segments treated with 2,4-dinitrophenol in the presence of indoleacetic acid or indoleacetic acid plus kinetin was remarkably higher than in corresponding segments which received no 2,4-dinitrophenol.     

Mechanism of a Synergistic Effect of Kinetin on Auxin-induced Ethylene Production: Suppression of Auxin Conjugation

In hypocotyl segments of mung bean (Phaseolus mungo L.) seedlings, exogenously supplied indoleacetic acid was rapidly conjugated mainly into indoleacetylaspartic acid, which was inactive in inducing ethylene production. Kinetin is known to stimulate indoleacetic acid-induced ethylene production. The mechanism of kinetin action on indoleacetic acid-induced ethylene production by hypocotyl segments of mung bean seedlings was studied in relation to indoleacetic acid uptake and indoleacetic acid metabolism. Kinetin enhanced indoleacetic acid uptake during the initial 2-hour incubation and markedly suppressed the conversion of indoleacetic acid to indoleacetic acid conjugates throughout the whole 7-hour incubation. As a result, there was more free indoleacetic acid and less conjugated indoleacetic acid in the segments treated with kinetin than in those receiving no kinetin. A close relationship was demonstrated between the rate of ethylene production and the level of free indoleacetic acid, which was regulated by kinetin.     

Tissue cultures of Artemisia pallens: Organogenesis,terpenoid production

Germinated seedlings of Artemisia pallens gave three types of cultures on MS medium supplemented with different plant growth hormones. Medium containing BA+2,4-D stimulated unorganized callus; BA+IAA medium, semi-organized tissues interspersed with shoot buds; and BA+NAA+IAA medium, multiple shoot cultures. The in vitro shoots developed roots in medium devoid of growth hormones. TLC and GLC analysis of the tissue extracts showed that linalool was present in the cultured tissues, with maximum concentration in the unorganized tissue. Although the TLC profiles of the three culture extracts were similar, the extracts did not contain the major polar compounds of the plant. The plant extracts contained more polar compounds and gave the characteristic fragrance of davana.Abbreviations MS Murashige & Skoog's basal medium - BA benzyladenine - Kn kinetin - NAA naphthaleneacetic acid - IAA indoleacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - PCV packed cell volume     

Hormonal control of isoperoxidases in lentil embryonic axis

Detachment of the cotyledons from the lentil (Lens culinaris Med.) embryonic axis causes in the latter an increase in total peroxidase activity which is shown to be due to enhancement of specific cathodic isoperoxidases. Kinetin treatment of attached or detached axes promotes activity of essentially the same cathodic isoperoxidases. In addition kinetin enhances the activity of two anodic peroxidases and represses specifically that of a cathodic one. Abscisic acid inhibits the production of all isoenzymes in the presence or absence of kinetin. Cytokinin and abscisic acid actions are discussed in relation to the nature of a wounding hormone and the role of natural inhibitors in cotyledons during germination. Indoleacetic acid stimulates the activity of certain isoenzymes which are also stimulated by kinetin, whereas in others the effects of the two hormones are different. Specific inverse effects of indoleacetic acid and kinetin are demonstrated on the two most cathodic isoperoxidases. Indoleacetic acid-kinetin interactions on the cathodic isoperoxidases have been found in the literature and are discussed as a possible mechanism for explaining interactions of the two regulators on growth and other physiological processes.     

Hormonal Control of Differentiation of Shoots, Roots and Embryos in Leaf and Stem Cultures of Petunia inflata and Petunia hybrida

Factors influencing adventitious bud and root development, callus induction and embryogenesis were investigated in stem and leaf cultures of Petunia inflata R. E. Fries and Petunia hybrida cv. Cascade and cv. Rose du ciel grown on a synthetic nutrient medium. Indoleacetic acid caused limited callus development and root formation whereas naphthaleneacetic acid Induced abundant roots. 2,4-Dichlorophenoxyacetic acid promoted callus growth and differentiation of embryos which eventually developed into plantlets. Cytokinins such as benzyladenine, zeatin and kinetin induced bud development. A combination of auxins and cytokinins caused an interaction which was manifested in altered morphogenetic response. Thus 2,4-dichlorophenoxyacetic acid in conjunction with benzyladenine caused suppression of bud development and retarded differentiation of embryos. Likewise, when benzyladenine was used with indoleacetic acid root development was totally inhibited and abundant buds were produced.     

Plant regeneration from tissue cultures of Persian buttercup (Ranunculus asiaticus L.)

Different plant explants of Persian buttercup (Ranunculus asiaticus L.) were screened for callus induction and adventitious shoot regeneration on different media to establish totipotent cultures. Murashige & Skoog (MS) medium was used, supplemented with different concentrations of the following growth regulators: kinetin, benzyladenine (BA), naphthaleneacetic acid (NAA) and indoleacetic acid (IAA). Callus was induced and adventitious buds regenerated only from cotyledonary explants after 4–5 weeks. Subculture of the regenerated buds on the same basal medium in presence of gibberellic acid (GA₃) and BA produced well-organized shoots. Rooting was obtained by transferring shoots to growth regulator-free MS medium. A high rate of shoot multiplication has been achieved on medium with high concentration of kinetin and long-day photoperiod. Finally the plants were successfully transferred to soil and grown in a greenhouse.     

Effect of phytohormones on plant regeneration in callus culture of <Emphasis Type="Italic">Iris ensata</Emphasis> Thunb

The effect of phytohormones on plant regeneration in callus culture of Iris ensata Thunb. was studied. 2,4-Dichlorophenoxyacetic acid (2,4-D) proved to be the most suitable auxin for the induction and subculturing of morphogenic callus. Organogenic calluses were induced from isolated embryos at the waxphase in MS medium supplemented with 2,4-D (1.0–2.0 mg/l) and kinetin (0.2–0.5 mg/l). Changes in the medium hormonal composition favor the development of adventitious structures. The setting of adventitious shoot buds took place in the presence of 6-benzylaminopurine, while the development of shoots and root primordia was observed after 2,4-D replacement with indoleacetic acid (2.0 mg/l). The root initiation in regenerating plants required a hormone-free medium. The development of intact seedling and regenerated plants of I. ensata were studied. Analysis of the shoot structure demonstrated that in vitro cultured plants are at the juvenile stage.     

A morphogenetic role for ethylene in hypocotyl cultures of Digitalis obscura L.

The effect of exogenously applied ethylene on organogenesis in Digitalis obscura L. hypocotyls cultured in vitro was studied. Interactions of this gas with other growth regulators was also tested. Ethylene by itself only promoted root formation. Shoot regeneration was obtained in presence of indoleacetic acid and kinetin. The addition of ethylene (10 ppm) increased the caulogenetic action of this medium; higher concentrations than 10 ppm reduced this response. Kinetin alone did not promote organogenesis and nullified the promotive effect of ethylene on rhizogenesis.Abbreviations BM basal medium - IAA indoleacetic acid - Kn kinetin     

Chemical Regulation of Callus Growth, Organogenesis, Plant Regeneration, and Somatic Embryogenesis in Antirrhinum majus Tissue and Cell Cultures

Excised stem explants of Antirrhinum majus L. var. ‘Kymosyblanc’ were grown in a denned medium to investigate factorsinfluencing bud and root development, callus induction, andsomatic embryogenesis. Auxins such as indoleacetic acid (IAA)and naphthaleneacetie acid (NAA) caused limited callus developmentand abundant root formation, whereas 2,4-dichlorophenoxyaceticacid (2,4-D) promoted soft friable callus with embryos and occasionaldevelopment of thick abnormal roots. 2-Naphthoxyacetic acid(NOA) and coconut milk (CM) used together induced friable greencallus growth and differentiation of small globular embryoswhich eventually developed into plantlets after transfer toauxin-free agar mineral medium containing sucrose. Cytokininssuch as benzyladenine (BA), zeatin, and kinetin induced compactgreen callus but in the absence of auxin failed to promote organogenesis.The interaction of IAA and kinetin resulted in the regenerationof the whole plant from stem explants. When NAA was used withkinetin, shoot development was totally inhibited and abundantroots were formed. Thus, the alternative morphogenetic eventsprobably reflect the biochemical subtleties occurring withinthe callus as a result of differences of actual endogenous levelsof growth substances in the tissues studied. These experimentshave been performed and interpreted on a histological basis.     

THE EFFECTS OF HORMONES UPON THE DEVELOPMENT OF EXCISED FLORAL BUDS OF AQUILEGIA

Young excised floral buds of Aquilegia were grown on defined medium containing kinetin, indoleacetic acid (IAA), or gibberellic acid (GA₃). Only when 10⁻⁶ or 10⁻⁷ m kinetin was added to the basal medium was there a significant increase in the number of initiated whorls of primordia. Buds on the basal medium or on medium with IAA or GA₃ failed to initiate carpels. On medium with 10⁻⁶ or 10⁻⁷ m kinetin, buds successfully initiated a normal whorl of five carpels. A high level of inorganic nitrogen was also required for the initiation of carpels. With 10⁻⁵ m kinetin, individual buds initiated from 6–18 carpels. Staminodial primordia of these buds were replaced with carpels, or the floral apex enlarged to accommodate a single whorl of many carpels. Kinetin did not support the further differentiation of the floral organs. Sepals, petals, and carpels did differentiate on medium with GA₃, but stamens aborted. However, on medium with GA₃ and kinetin, stamen primordia differentiated into short filaments and anthers. Further unknown growth factors appear to be required for the complete differentiation of floral primordia into mature organs.     

Somatic embryogenesis from hypocotyl callus cultures of Digitalis obscura L.

Hypocotyl-derived calli obtained in agar solidified medium with several growth regulator combinations gave rise to proembryonal masses and globular embryos when transferred to liquid media with lower growth regulator and higher NH₄HO₃ levels. By transferring cultures from liquid media to different solidified media, new embryo formation took place, but further development of these embryos or those previously induced depended on the characteristics of these media. Normal development was only achieved on 8 g/l agar solidified medium without growth regulators. Typical cotyledonary embryos developed into whole plants when transferred to this same medium.Abbreviations BA 6-benzylaminopurine - CH casein hydrolysate - CM coconut milk - 2,4-D 2,4-dichlorophenoxyacetic acid - 2iP 2-isopentenyladenine - Kn kinetin - NAA naphthaleneacetic acid - IAA indoleacetic acid     

Morphogenic Potentialities of Flower Buds of Kalanchoe pinnata Pers. grown in vitro

Excised flower buds of Kalanchoe pinnata Pers. representingtwo early stages of development (designated sets I and II),were cultured on modified White's medium (WB). They failed toattain full development on WB or on WB containing any of thefollowing supplements: indole-3yl-acetic acid (IAA), 2,4-dichlorophenoxyaceticacid (2,4-D), naphthaleneacetic acid (NAA), kinetin, or coconutmilk (CM). A slight stimulation of the growth of corolla wascaused by kinetin (10 ppm). IAA (I ppm) and NAA (I ppm) inducedrooting from the cut end of the pedicel and from the proliferatedtorus tissue situated between the sepals and petals. 2,4-D (Ippm) either singly or in concert with CM (10 per cent) stimulatedthe formation of shoot buds and root growth. Addition of kinetin(I and 10 ppm) to WB favoured shoot formation, but suppressedrooting. Flower buds of set II developed shoot buds more readilythan those of set I. Thus, the primordia floral organs presentin the immature buds lose their ability for normal morphogenesisunder culture conditions. Buds destined to form flowers canbe made to revert to vegetative growth.     

Induction of adventitious buds in cultured petal explants ofChelidonium majus L.

Petal explants ofChelidonium majus L. (Papaveraceae) formed noteworthy adventitious buds without any intermediate callus when cultured under appropriate conditions. Bud formation was favored by combinations of 1–2 mg/l indoleacetic acid (IAA) and/or 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1–0.5 mg/l kinetin (K). In the present study, neither bud formation nor callus formation occurred in cultures of excised leaves. A histological study revealed that adventitious bud formation occurred only in single epidermal layers of petals, while several subepidermal parenchyma layers did not join in its formation. Activation zones arising from the epidermis underwent intense cell divisions to initiate buds on the epidermal surface. These buds later turned green in color, developing into shoots which eventually grew into plantlets after root formation.     

Cytokinin-controlled Indoleacetic Acid Oxidase Isoenzymes in Tobacco Callus Cultures

Indoleacetic acid oxidase in tobacco callus tissues (Nicotiana tabacum L., cultivar White Gold) was resolved into seven anionic isoenzymes by polyacrylamide gel disc electrophoresis. Different concentrations of kinetin and zeatin in the presence of indoleacetic acid affected the level of this enzyme, particularly two fast-moving isoenzymes, A₅ and A₆. The optimal concentration of kinetin was 0.2 μm; increasing concentrations above this level progressively lowered the total activity of indoleacetic acid oxidase and repressed the development of isoenzymes A₅ and A₆. Actinomycin D and cycloheximide inhibited the development of these two isoenzymes under the influence of 0.2 μm kinetin, suggesting a requirement for RNA and protein synthesis. The cytokinin-promoted indoleacetic acid oxidase isoenzymes A₅ and A₆ increased with time and paralleled the dry weight increase of tobacco callus tissues, but the total activity of indoleacetic acid oxidase per unit dry weight of tobacco callus varied with time depending on the stage of plant growth.     

Plant regeneration from Bulgarian rose callus

Plant regeneration capacity of Bulgarian rose callus tissue was examined. Adventitious bud formation could be successfully attained, depending on the kinds of mineral salts used in the medium, auxin and cytokinin used. When callus tissues were cultured on the medium without ammonium nitrate and contained indoleacetic acid and benzyladenine, buds were formed in the callus. The number of buds were significantly increased by the simultaneous addition of calcium ionophore. When the cultures were transferred to the medium without cytokinin, roots were formed in the basal part of the buds.Abbreviations BA benzyladenine - IAA indoleacetic acid - K kinetin - NAA naphthaleneacetic acid