Induction and development of somatic embryos from spinach (Spinacia oleracea) leaf segments

Somatic embryogenesis was induced in embryogenic calli of Spinacia oleracea L., on a Murashige and Skoog (1962) medium, containing 4.6 μM kinetin as the sole growth regulator. Abscisic acid, gibberellic acid, and indole-3-acetic acid were supplemented to kinetin-containing medium and their effects on the initiation of somatic embryos was studied. Abscisic acid at a particular concentration (4 μM) dramatically increased the number of embryos per g fresh weight of callus, while both gibberellic acid and indole-3-acetic acid suppressed the embryo initiation. It is suggested that the promoting effect of abscisic acid on the embryo initiation may be explained as a stress response of the tissue. The relative number of globular embryos vs. the embryos in heart/torpedo and cotyledonary stages was increased at 4 μM abscisic acid and at all gibberellic acid concentrations (0.3–10 μM). In contrast, the ratio of globular to polar embryos was lower than in controls at 1 μM abscisic acid and indole-3-acetic acid (1 and 10 μM). The effects of growth regulators on the ratio of globular to polar embryos indicate that they interfere with the normal distribution of cell division and cell expansion during early embryogenesis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Micropropagation of a fruit tree, Morus australis Poir. syn. M. acidosa Griff.

High frequency bud break and multiple shoots were induced in nodal explants collected between November to February from a 5 year old tree of Morus australis Poir syn. M. acidosa Griff. on Murashige and Skoog's medium supplemented with 6-benzylaminopurine (1.0 mg/1). Incorporation of gibberellic acid (0.3 mg/l) along with BAP (1.0 mg/l) not only induced faster bud break from nodal explants as well as from apical shoot buds, but it also enhanced the frequency of bud break. Nodal explants were more responsive than apical shoot buds. The shoots formed in vitro were multiplied further as nodal segments, and an average multiplication rate of 6-fold per subculture was established within 4–5 months. The shoots were successfully rooted on half-strength MS containing a combination of indole-3-acetic acid, indole-3-butyric acid and indole-3-propionic acid, each at 1.0 mg/1. The plantlets were successfully hardened off and established in natural soil.Abbreviations BAP 6-benzylaminopurine - GA₃ gibberellic acid - KN kinetin - IAA indole-3-acetic acid - IBA indole-3-butyric acid - IPA indole-3-propionic acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthalene acetic acid     

Anthocyanin production in callus cultures of roselle (Hibiscus sabdariffa L.)

Callus tissues derived from seedlings of roselle (Hibiscus sabdariffa L.) were shown to produce two cyanidin glycosides as major anthocyanin pigments. Both callus growth and anthocyanin synthesis were remarkably stimulated by 2,4-dichlorophenoxyacetic acid. The highest anthocyanin yield was observed when 1 M 2,4-D in combination with 0.1–1 M kinetin was supplemented to the culture medium. In contrast, gibberellic acid showed inhibitory effect on anthocyanin production.Abbreviations LS Linsmaier and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - GA gibberellic acid     

High efficiency plant regeneration from cotyledons of watermelon (Citrullus vulgaris Schrad)

Cotyledons of various ages from seedlings of eight watermelon (Citrullus vulgaris) cultivars were cultured on MS medium supplemented with different combinations of phytohormones. High frequency shoot regeneration (60.0–92.0%) was induced from 5-day-old cotyledons of cultivars cultured on MS medium containing 5.0 mg/l 6-benzylaminopurine (BA) and 0.5 mg/l indole-3-acetic acid (IAA). Multiple shoot buds elongated on MS medium containing 0.2 mg/l kinetin (KT) and 5–10 shoots per expiant could be recovered depending on the cultivars. Elongated shoots rooted on MS medium with 0.1 mg/l -naphthalene acetic acid (NAA). Zeatin riboside (ZT) had a similar efficiency as BA in shoot induction, and it was significantly more functional than 2-isopentenyladenine (2iP) or kinetin (KT). Cotyledons from 5-day-old seedlings were the most responsive to shoot induction.Abbreviation BA 6-benzylaminopurine - GA₃ gibberellic acid - IAA indole-3-acetic acid - 2iP 2-isopentenyladenine - KT kinetin - MS Murashige and Skoog (1962) - NAA -naphthalene acetic acid - ZT zeatin riboside     

Phytohormone effects on hydrolytic activity of phosphohydrolases in red beet (Beta vulgaris L.) tonoplasts

The effects of indole-3-acetic acid (IAA), abscisic acid (ABA), gibberellic acid (GA₃) and kinetin on the hydrolytic activity of proton pumps (adenosine triphosphatase, H⁺-ATPase, pyrophosphatase, H⁺-PPase) of tonoplasts isolated from stored red beet (Beta vulgaris L. cv. Bordo) roots were studied. Results suggest that the phytohormones can regulate the hydrolytic activities of H⁺-ATPase and H⁺-PPase of the vacuolar membrane. Each of the proton pumps of the tonoplast has its own regulators in spite of similar localization and functions. IAA and kinetin seem to be regulators of the hydrolytic activity for H⁺-PPase whereas for H⁺-ATPase it may be GA₃. Stimulation of enzyme activity by all hormones occurred at concentrations of 10^–6 to 10^–7 M.Abbreviations IAA indole-3-acetic acid - ABA abscisic acid - GA₃ gibberellic acid - H⁺-ATPase adenosine triphosphatase - H⁺-PPase pyrophosphatase - ATP adenosine triphosphate - Tris Tris (hydroxymethyl)-aminomethane - MES (2[N-Morpholino]) ethane sulfonic acid - EDTA ethylene diamine tetraacetic acid - P_i inorganic phosphate     

The effect of selected plant hormones on in vitro proliferation of hyphae of Glomus fistulosum

Effects of N⁶-benzyladenine, kinetin, zeatin, N⁶-benzyladenosine, kinetin riboside, zeatin riboside, jasmonic acid, indole-3-acetic acid, indole-3-butyric acid, indole-3-propionic acid, abscisic acid and gibberellic acid on proliferation of hyphae of arbuscular mycorrhizal (AM) fungus Glomus fistulosum were studied under axenic conditions in vitro. The growth of intraradical hyphae of G. fistulosum was fully suppressed by 30 μM indole-3-acetic acid, but a perceptible decrease in the proliferation of the hyphae was observed already at 3 μM. Because such concentration is near the concentrations common in root tissues in vivo, the effect may be biologically significant. Similar effect was also observed for Glomus mosseae. Inhibitory effects of abscisic acid and cytokinins occurred only at very high, non-physiological concentrations. Ribosylated cytokinins showed stronger inhibition effects than their non-ribosylated counterparts. No stimulation of proliferation of hyphae by any plant hormone tested was observed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Morphogenetic potential of foliar explants in Duboisia myoporoides R.Br. (Solanaceae)

The effects of serial combinations of either indole-3-acetic acid, indole-3-butyric acid or -naphthaleneacetic acid (0.5–10.0 mg/l) with either kinetin, 6-benzyl-amino-purine, zeatin or 6-methylaminopurine (0.5–5.0 mg/l) have been investigated to assess the morphogenetic potential of foliar explants of Duboisia myoporoides. Shoot buds developed either directly or via a callus interphase. Combinations involving indole-3-acetic acid with any of the cytokinins were more effective in inducing shoot bud formation compared to those containing indole-3-butyric acid or -napthalenacetic acid as an auxin. Among cytokinins, zeatin, kinetin and 6-benzylamino-purine were equally effective for shoot formation. However, optimum response with zeatin could be achieved at low concentrations (0.5–2.0 mg/l), while kinetin and 6-benzylamino-purine exhibited comparable efficacy at higher levels (3.0–5.0 mg/l). 6-Methylaminopurine proved least effective in all concentrations and combinations tested. Rooting of the differentiated shoots was readily achieved with -naphthaleneacetic acid alone (0.5 mg/l) after changing the physical form of the medium from gel to static liquid. Regenerated plantlets were transferred to pots and grown to maturity in the field with a high rate of survival (80–90%).Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MAP 6-methylaminopurine - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic acid - PVP polyvinyl pyrrolidone     

Synergism between Gibberellin and Auxin in the Growth of Intact Tomato

Gibberellin A_4&7 was more effective than gibberellic acid in increasing shoot elongation when applied to the apex of intact Lycopersicum esculentum seedlings of Tiny Tim, a dwarf cultivar, and Winsall, a tall cultivar. After 14 days, gibberellic acid and gibberellin A_4&7 stimulated growth of the dwarf more than the tall tomato. In tall tomato the application of indole-3-acetic acid alone (6.1 μg/plant) showed an inhibitory growth effect, but when applied with 17.5 μg per plant of gibberellic acid, it had a synergistic effect at 7 days but not at 14 days. When the auxin concentration was reduced to 0.61 μg per plant a synergistic effect was observed on tall plants at 7 and 14 days between indole-3-acetic acid and gibberellic acid. Application of gibberellin A_4&7 with auxin did not give a synergistic response in tall or dwarf tomato.     

Somatic embryogenesis from chickpea (Cicer arietinum L.) inmature cotyledons: The effect of zeatin, gibberellic acid and indole-3-butyric acid

Studies were conduced to test the effects of various cytokinins on somatic embryogenesis from chickpea (Cicer arietinum L.) immature cotyledons. Zeatin (13.7 μmol) added, to B5 basal medium, supplemented with 1.5 % sucrose and 0.2 μmol indole-3-acetic acid, was the most effective cytokinin. Lobular structures obtained from cotyledons cultures were transferred to B5 basal medium supplemented with gibberellic acid and indole-3-butyric acid at different concentrations. The most effective treatment was B5 medium containing 14.4 μmol gibberellic acid plus 1.0 μmol indole-3-butyric acid in which 42.8 % of lobular structures cultured formed normal somatic embryos. High conversion of embryos into plantlets (61.0–65.2 % embryos regenerated plants) was observed when germinated embryos were placed on plant development medium.     

The Effect of Growth Hormones on Cell Division and Expansion in Liquid Suspension Cultures of Acer pseudoplatanus

The effects on cell division and cell size of indole-3-aceticacid (IAA), gibberellic acid (GA), and kinetin were studiedin liquid suspension cultures of cambial cells derived fromAcer pseudoplatanus. It was shown that all three hormones promotecell division and that the effects of both GA and kinetin areadditive to those of IAA, but the effects of GA and kinetintogether are not additive. Treatment with IAA resulted in anincrease of mean cell size (indicating that cell expansion ispromoted), but after GA or kinetin treatment the mean cell sizewas smaller, indicating that little cell expansion had takenplace after each division. The results are discussed in relationto previous work on the effects of hormones in the intact cambiumand to current theories on the interactions of growth hormones.     

Micropropagation of Madhuca longifolia (Koenig) MacBride var. latifolia Roxb.

Bud break and multiple shoots were induced in apical and axillary meristems derived from 10-d old seedlings of Madhuca longifolia var. latifolia on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l N⁶-benzyladenine (BA) singly or in combinatiobn with 1-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA). Excised shoots were rooted on half-strength MS with IBA (1.0 mg/l) after 18d of culture. Regenerated plantlets were acclimatized and successfully transferred to soil.Abbreviations BA N⁶ benzyladenine - KN kinetin - ADS adenine sulphate - IBA indole-3-butyric acid - IAA indole3-acetic acid - NAA 1-naphthaleneacetic acid - MS Murashige and Skoog (1962) medium     

Role of Ascorbic Acid in Bud Development

Ascorbic acid was found to increase bud development in Pisum sativum L. The interactions of ascorbic acid with indole-3-acetic acid, kinetin and gibberellic acid were studied. It was found that ascorbic acid promoted bud growth and overcame the inhibitory effect of auxin. When applied with gibberellin, bud growth was greatly enhanced. Ascorbic acid promoted bud development in red light only; it did not in far-red or dark.     

Effects of ascorbic acid on axillary shoot induction in Tylophora indica (Burm. f.) Merrill.

A procedure for rapid in vitro multiplication of Tylophora indica (Burm. f.) Merrill., an important indigenous medicinal plant, has been developed. Addition of ascorbic acid was essential to induce sprouting of axillary buds. Optimum multiplication was observed on MS medium containing 6-benzylamino purine (5.0 mg l^–1), -naphathalene-acetic acid (0.5 mg l^–1) and ascorbic acid (100 mg l^–1). Rooting of in vitro produced shoots was readily achieved with indole-3-acetic acid alone (1.0 mg l^–1) in MS. The plantlets thus obtained were successfully transferred to pots in large numbers which grew normally.Abbreviations BAP 6-benzylamino purine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2ip 2-isopentenyladenine - Kn kinetin - MS Murashige & Skoog media - NAA -naphthalene acetic acid     

Enhancement of growth and chitosan production by Rhizopus oryzae in whey medium by plant growth hormones

The effect of some plant growth hormones, viz., gibberellic acid, indole-3-acetic acid, indole-3-butyric acid, and kinetin on chitosan production by Rhizopus oryzae in deproteinized whey was studied. Hormones, at different concentrations, increase the mycelial growth by 19-32%. However, increase in chitosan content of the mycelia was relatively small (1.7-14.3%) over the control. Maximum enhancement was observed with gibberellic acid. Fifty percent more chitosan could be obtained from 1L of whey containing 0.1mg/L gibberellic acid. Hormones, at higher dose, instead of stimulation inhibited both growth and mycelial chitosan content. This study showed that hormones have no influence on degree of deacetylation of chitosan but increase the quality of the chitosan by increasing weight average molecular weight and decreasing polydispersity. All the hormones had been found to enhance chitin deacetylase activity of R. oryzae by 1.067-1.267-fold and may be one of the reasons for increased chitosan production.     

Regeneration of Picea omorika plants via organogenesis

Picea omorika plants were regenerated from embryo and seedling shoot tip cultures. Adventitious and axillary shoots were produced on 1/2 MS medium containing benzyladenine and kinetin. Benzyladenine was more effective in bud induction, whereas kinetin hastened shoot development. Excised shoots were elongated on 1/3 MS medium without growth regulators, multiplied with kinetin and rooted with or without indole-3-butyric acid.Abbreviations BA N⁶-benzyladenine - 2IP N ⁶-(²-isopenteny) adenine - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid     

Adventive plants from ovules and nucelli in Citrus

Summary 1- to 8-week-old ovules and nucelli from three Citrus cultivars—Shamouti and Valencia (Citrus sinensis) oranges and Marsh Seedless (C. paradisi) grapefruit—were cultured in vitro. No embryo differentiation was observed in the explants prior to culture. The Shamouti ovules had degenerated and were apparently unfertilized. Embryoids formed on Murashige and Tucker nutrient medium supplemented with 500 mg/l malt extract. Whole plants developed on the same basal medium supplemented with kinetin and indole-3-acetic acid (IAA), coconut milk or gibberellic acid (GA₃). A higher kinetin/IAA ratio or the addition of coconut milk favoured stem elongation more than root formation while a lower kinetin/IAA ratio favoured root formation and inhibited stem elongation. The addition of GA₃ to the basal medium stimulated rooting and stem elongation. These results can be of aid in mutation research, allowing irradiation at stages prior to embryonic development.     

Growth and Cellular Differentiation in the Wheat Coleoptile (Triticum vulgare): II. FACTORS INFLUENCING THE GROWTH RESPONSE TO GIBBERELLIC ACID, KINETIN AND INDOLYL-3-ACETIC ACID

The sequential growth response of excised wheat coleoptilesduring growth and cellular differentiation to solutions of gibberellicacid, kinetin, and indolyl-3-acetic acid is further analysedin this paper. Excised yound coleoptiles which possess a low potential growth-rate(controls) respond to gibberellic acid and kinetin, whereasexcised older coleoptiles which have a high potential growth-raterespond to indolyl-3-acetic acid. The difference between theseresults and those reported for pea-stem sections is discussed. To obtain a well-defined separation of the sequential growthresponse to gibberellic acid and kinetin it is necessary togrow the wheat coleoptiles at 22.5°C. At a higher temperature(25°C) the two optimal responses tend to overlap. It issuggested that this difference is due to a delayed onset ofcell division at the lower of the two temperatures. Substances diffusing from the primary leaves contained withinthe excised coleoptile do not interfere with the sequentialgrowth response of the coleoptile. Differential uptake of thegrowth regulators into the older coleoptiles does not explainthe lower growth response stimulated in these coleoptiles bygibberellic acid and kinetin as compared to the high activityof indolyl-3-acetic acid. The possible importance of gibberllic acid in primary cell-wallgrowth is discussed.     

Micropropagation of lemon balm

Traditional propagation of lemon balm (Melissa officinalis L.) is inefficient for establishing a good quality clonal population. Results of the presented experiments outline an effective method for micropropagation of this species. Following culture initiation from shoots of field-grown plants on growth regulator free Murashige–Skoog medium, rapid shoot multiplication with only rudimentary root formation could be achieved on media containing various concentrations of indole-3-acetic acid and 6-benzyladenine. The combination of 5.71 μM indole-3-acetic acid and 6.66 μM 6-benzyladenine resulted in the best multiplication. Transfer of propagules to media containing indole-3-acetic acid and kinetin did not result in shoot proliferation; however, single plantlets grown on media containing 5.71 μM indole-3-acetic acid and 13.9 μM kinetin developed more compact shoots and stronger roots than the control plants and were suitable for acclimatisation with an efficiency over 95%. This revised version was published online in June 2006 with corrections to the Cover Date.     

Studies on plant regeneration from cotyledonary protoplasts in Brassica campestris

Protoplasts isolated from both 7-day-old light-grown and 4-day-old dark/dim light-grown cotyledons of four Brassica campestris varieties (Arlo, Sonja, Bunyip and Wonk Bok) were cultured in three liquid media: modified K8P, modified MS and modified Pelletier's B to compare the capacities for cell division and plant regeneration. Following cell wall regeneration the cultured protoplasts from dark/dim light-grown cotyledons of four varieties showed rapid division and high frequency of cell division compared with those isolated from light-grown cotyledons. The frequencies of cell division were significantly influenced by varieties and culture media but only in cultured protoplasts isolated from dark/dim light-grown cotyledons. The interaction between varieties and media was also significant. Cell colonies formed within 7–14 days in protoplast cultures from dark/dim light-grown cotyledons, and calli subsequently grown on a solid medium developed shoots when transferred onto a regeneration medium. Three of four tested varieties (Arlo, Sonja and Bunyip) showed shoot regeneration within 2–3 months after protoplast isolation, with a high degree of reproducibility in Arlo and Bunyip. Regenerated shoots, which were induced to root on half-strength MS medium with 0.1 mg.l^–1 IBA, survived in soil and grew to produce siliques and set viable seeds in the greenhouse. The present report is the first to document the production of regenerated plants that set seeds in Brassica campestris from cotyledonary protoplasts.Abbreviations BAP benzylaminopurine - CPW Composition of Protoplast Washing-solution - 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylenediamine-tetraacetic acid - GA₃ gibberellic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - KT kinetin - FDA fluorescein diacetate - SDS sodium dodecyl sulfate     

Alleviation of NaCl Stress by Pretreatment with Phytohormones in Vigna radiata

Efficiency of pretreatment as foliar spray of indole-3-acetic acid, gibberellic acid and kinetin, each ranging from 0.1 to 10.0 μM concentration, in restoring the metabolic alterations imposed by NaCl salinity was investigated in Vigna radiata (L.) Wilczek. Glycolate oxidase, superoxide dismutase, catalase and peroxidase activities increased under stress in leaves and roots also. Malondialdehyde content and total peroxide content also increased under stress. All the three hormones used were able to overcome to variable extents the adverse effects of stress imposed by NaCl to these parameters. This revised version was published online in July 2006 with corrections to the Cover Date.