Effects of gibberellic acid and zeatin on the growth response of cucumber cotyledons

In contrast to cytokinin, gibberellic acid has no effect on the growth of the isolated cucumber cotyledon in darkness. Like cytokinins in light, gibberellic acid causes increases in fresh weight and area of the cotyledon at concentrations from 10^–7 to 10^–3 M. Radiant energies in the blue, red, and far-red regions of the spectrum all induce the growth responses to gibberellic acid. The effect of the far red is greater than that of the red, which is greater than that of the blue. Gibberellic acid is ineffective in the promotion of chlorophyll development, whereas cytokinins are very effective. Although zeatin and gibberellic acid both cause an increase in fresh weight and area of the cotyledons in light, they appear to have entirely separate actions in the growth responses.     

Organic nitrogen stimulates caulogenesis from hypocotyl callus of Phyllanthus fraternus

Factors involved in promoting caulogenesis from hypocotyl explants of Phyllanthus fraternus were studied. Hypocotyl explants were cultured on B₅ medium supplemented with 2,4-D or NAA in the presence and absence of BAP (at concentrations 0, 10^–7, 10^–6 and 10^–5M). Adventitious shoots differentiated from callus developed from the cut ends of 12.5% of the hypocotyl segments cultured on medium supplemented with 10^–6M BAP in combination with 10^–6M 2,4-D or 10^–6M NAA. Profuse rooting occurred from the hypocotyl explants on medium supplemented with 10^–6M BAP + 10^–6M NAA. Incorporation of casein hydrolysate in B₅ medium along with 10^–6M BAP + 10^–7M 2,4-D enhanced the frequency of cultures with adventitious shoots upto 68.0%. Glutamine, glutamic acid or proline could partially substitute for the effect of casein hydrolysate. Amongst the hypocotyls from 3–14 d old seedlings, the best caulogenesis was obtained with hypocotyls from 7 d old seedlings both in presence or absence of casein hydrolysate. Best rooting of shoots was achieved on half-strength B₅ medium supplemented with 10^–6M IBA. After hardening, plantlets were successfully transferred to the soil.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2, 4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - CH casein hydrolysate - Arg L-arginine - Glu L-glutamic acid - Gln L-glutamine - Leu L-leucine - Lys L-lysine - Pro L-proline     

Micropropagation of sugarcane (Saccharum spp.)

A method for callus induction, adventitious bud regeneration, shoot multiplication and rooting of in vitro formed shoots of Helianthus annuus L. var. Argentario is described. Hypocotyl and cotyledon explants formed callus on medium containing 2 mgl^–1 naphthalene acetic acid and 0.5 mgl^–1 benzyladenine. Adventitious buds were formed on hypocotyl segments on medium containing 0.5–2 mgl^–1 benzyladenine. The optimal level of sucrose concentration for shoot regeneration from hypocotyls was 1.5%. Multiplication from shoot apices was promoted by kinetin (2 mgl^–1) plus gibberellic acid (5 mgl^–1), benzyladenine (2 mgl^–1) plus gibberellic acid (10 mgl^–1) or at lower frequency by benzyladenine (1 mgl^–1). A general feature of the plantlets formed in vitro was the precocious flowering.     

Cytokinins promotion of flowering in Cymbidium ensifolium var. misericors in vitro

Callus-derived rhizomes of Cymbidium ensifolium var. misericors produced flowers precociously on a defined basal medium (1/2MS) containing of NAA with thidiazuron (TDZ), N⁶-(2-isopentenyl) adenine (2iP) or N⁶-benzyladenine (BA) within 100 d of culturing. Among eight cytokinins tested, TDZ at 3.3–10 µM or 2iP at 10–33 µM combined with 1.5 µM NAA were the most effective combinations for achieving flower induction in vitro. These undersized flowers were physically normal and bloomed for two weeks in vitro.     

Rapid in vitro differentiation of Sesbania bispinosa plants — a leguminous shrub

Multiple shoots differentiated from hypocotyl explants of Sesbania bispinosa (Jacq.) W.F. Wight, a leguminous woody shrub, when cultured on Gamborg's basal medium alone or in combination with 6-benzyl aminopurine (10^–7–10^–4 M). For cotyledonary explants 6-benzyl aminopurine (10^–6–10^–4 M) was necessary. The shoots rooted when cultured on Gamborg's basal medium containing indole-3-butyric acid (10^-5 M). Plantlets thus formed were transferred to soil where they have flowered and also set fruits.     

Regeneration of multiple shoots and plants from Mesembryanthemum crystallinum

Mesembryanthemum crystallinum plants have been regenerated via organogenesis from hypocotyl, cotyledonary node, and leaf expiants with varying frequencies. The highest regeneration frequencies were obtained from either hypocotyls (23–34%) or cotyledonary nodes (21–41%). Leaf expiants yielded very poor regeneration frequencies (0–11%). Expiants were placed on Murashige and Skoog (MS) media supplemented with 3% sucrose, 0.8% bacto-agar and either, 10.8×10^–6M NAA and 8.8×10^–6M BA (MSmsh), 1×10^–5M BA and 1×10^–6M IAA, (MS4) or 1×10^–6M BA and 1×10^–6M IAA (MS5). Shoot formation frequencies were greater on MS4 and MS5 and lower on MSmsh, however, overall differences of regeneration frequency among media tested were not statistically significant. Regenerated plantlets were rooted on MS medium without growth regulators. Mature, regenerated plants were fertile and exhibited DNA content and ploidy profiles that were identical to wild type plants.Abbreviations MS Murashige and Skoog media - CAM Crassulacean acid metabolism - kbp kilobase pairs - NAA 1-naphthaleneacetic acid - BA 6-benzyladenine - IAA indole-3-acetic acid     

Regeneration of plants from primary leaves of cowpea

Plants were regenerated from the in vitro cultured explants of primary leaves of cowpea (Vigna unguiculata L. Walp). Primary leaves, including the intact petiole, were excised from three-day-old seedlings and cultured on Gamborg's B5 basal medium containing 8×10^–7 M 2,4,5-trichlorophenoxyacetic acid, 1×10^–2 M L-glutamine and 1×10^–4 M adenine sulfate. Callus formed at the petiole end. Prolific shoot regeneration occurred when this callus was transferred to B5 basal medium containing 5×10^–6 M 6-benzyl-aminopurine (BAP). Regenerated shoots rooted in growth-regulator-free B5 basal medium and were established in soil.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - NAA 1-napthalene acetic acid - 2,4,5-T 2,4,5-trichloro-phenoxyacetic acid     

Regeneration of plants from callus cultures of Origanum vulgare L.

Investigations were undertaken to achieve rapid multiplication and improvement of Origanum vulgare (a herbaceous, ornamental plant well known for its aromatic and medicinal value) through plant regeneration from callus. The explants (cotyledons, hypocotyl and root segments) excised from 15 d old aseptic seedlings were cultured on Gamborg's B₅ medium supplemented with 2,4-D, NAA and BAP individually and in various combinations (at concentrations of 0,10^–7,10^–6 and 10^–5 M). Best callus induction was noted on medium with 10^–7 M 2,4-D alone. The cotyledonary expiants proved to be the best source for compact and nodulated callus. The subcultured cotyledonary calli showed shoot induction when transferred onto media supplemented with BAP alone orin combination with 10^–7M or 10^–6MNAA. However, 10^–5M NAA completely suppressed the shoot inducing ability of BAP. In general, NAA promoted root induction from all explants used including cotyledonary callus. Best shoot induction was obtained on medium supplemented with 10^–6M BAP+10^–6MNAA. Both IBA and NAA at 10^–6 M proved to be equally effective in induction of roots from the cut ends of 15–20 mm long shoots (excised from callus) in half-strength B₅ liquid medium. Rooted shoots were successfully re-established in soil under controlled conditions.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine     

A tissue culture of the Ostrich fern Matteuccia struthiopteris (L.) Todaro

Multiple bud formation was induced from shoot apices of Matteuccia struthiopteris cultured on semi-solid Knudson's medium supplemented with 10^-5 and 10^-6 M kinetin. The effect of kinetin, naphthaleneacetic acid and gibberellic acid on shoot and root development is discussed and a three-part tissue culture system was devised for micropropagation and rooting.     

Effect of exogenous hormones on leaf yellowing in cut flowering branches of Alstroemeria pelegrina L.

Inclusion of IAA in the vase water had little effect on leaf yellowing in cut flowering branches of Alstroemeria pelegrina L. while kinetin delayed leaf yellowing at 10^–4M (continuous treatment). Chlorophyll was effectively retained by 10^–7M gibberellic acid (GA) in the vase water or by a 20h pulse at 5°C with 10^–5/10^–4M GA. After 16h of ¹⁴C-GA₃ uptake at 20°C relatively high levels of ¹⁴C were found in leaves and low levels in stems and flowers. After this treatment about half of the ¹⁴C-GA₃ in leaves was metabolized into unknown compounds.     

In vitro high frequency plant regeneration of a tree legume Tamarindus indica (L.)

Optimal culture conditions for high frequency plant regeneration from excised cotyledons of Tamarindus indica were established. Maximum shoot bud differentiation (100%) occurred when the adaxial surface of the entire cotyledon (excised from 12-d old seedlings) was in contact with MS medium containing 5×10^–6M BAP. On MS alone only roots were formed. Shoot or root formation was confined to nodal tissue at the top of the notch present on the adaxial surface at the proximal end of the cotyledon. Thirty-four to 95 shoots were regenerated in a 4 month period from individual cotyledons. Shoots were rooted on MS with 5.7×10^–6M IAA. IAA (5.7×10^–7M) alone induced complete plant formation. Regenerated plants were established in the soil with 70% success.Abbreviations BAP 6-benzylaminopurine - KIN kine-tin - 2-iP 6-Y-Y-dimethylallyl aminopurine - AdS adenine sulphate - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA 1-naphthalene acetic acid - MS Murashige and Skoog (1962) medium     

Plant regeneration of Solanum lycopersicoides Dun. from stem explants,callus and suspension cultures

Protocols were established for achieving plant regeneration from stem internode, callus, and cell suspension cultures of Solanum lycopersicoides Dun. Two accessions of S. lycopersicoides exhibited different responses as to callus formation on various media, requirement of gibberellic acid for shoot regeneration, and ability to grow in suspension culture. The optimum medium for initiation and maintenance of cell suspension cultures was Murashige and Skoog [9] medium with 15 mg l^– NAA. For shoot regeneration, of three cytokinins tested, zeatin was found most effective relative to number, rapidity of response and overall quality of shoots. Shoot regeneration from stem explants, callus and suspension cultures was optimum on MS + 3.0 mg l^–1 zeatin + 0.1 mg l^–1 gibberellic acid.Michigan Agricultural Experiment Station Journal Article No. 11589.     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

Cytokinins of the Developing Mango Fruit : Isolation, Identification, and Changes in Levels during Maturation

The cytokinin activity has been isolated and identified from extracts of immature mango (Mangifera indica L.) seeds. The structures of zeatin, zeatin riboside, and N⁶-(Δ²-isopentenyl)adenine riboside were confirmed on the basis of their chromatographic behavior and mass spectra of trimethylsilyl derivatives. Both trans and cis isomers of zeatin and zeatin riboside were also identified by the retention times of high performance liquid chromatography. In addition, an unidentified compound appeared to be a cytokinin glucoside.

The concentration of cytokinins in the panicle and pulp of mango reached a maximum 5 to 10 days after full bloom and decreased rapidly thereafter. The cytokinin level in the seed remained high until the 28th day after full bloom. The quantity of cytokinins in pulp per fruit increased from the 10th day after full bloom, the maximum being attained around the 50th day after full bloom. Similarly, the amount of cytokinins per seed increased from the 10th day after full bloom, reaching a peak on the 40th day and decreasing gradually thereafter.

A high percentage of fruit set in mango was persistently maintained by supplying 6-benzylaminopurine (1.5 × 10³ micromolar) onto the panicle at the anthesis stage and by supplying gibberellic acid (7.2 × 10² micromolar) and naphthalene acetamide (3.1 × 10 micromolar) at the young fruit stage.

     

Formation of plantlets through somatic embryogeny in the Himalayan blue poppy,Meconopsis simplicifolia (Papaveraceae)

Summary Exuberant and subculturable calli could be induced from only hypocotyl and leaf segments of ca 4-month-old seedlings of Meconopsis simplicifolia cultured on Murashige & Skoog's medium supplemented with 10^–6M kinetin + 10^–5M -naphthalene acetic acid. Suspension cultures were initiated from the calli in a similar medium but with 10^–5M 2,4-dichlorophenoxy acetic acid in place of -naphthalene acetic acid. In ca 80% of the suspension cultures somatic embryos differentiated freely (80–85%) as well as on the surface of small clumps of tissue (15–20%). Somatic embryos that developed beyond heart-shaped stage were transferred to agar-solidified Murashige & Skoog's medium free of growth substances. When maintained in 10 h light and 14 h dark the somatic embryos developed into plantlets bearing cauline leaves. From seed sowing to raising normal plantlets via callus required 28 weeks; on average 80 plantlets were obtained from one explant in three passages.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - FAA formalin-acetic acid-alcohol - GA₃ gibberellic acid - Kn kinetin - MS Murashige & Skoog's medium (Murashige and Skoog 1962) - NAA -naphthalene acetic acid     

Responsiveness of Amaranthus retroflexus seeds to ethephon, 1-aminocyclopropane 1-carboxylic acid and gibberellic acid in relation to temperature and dormancy

Dormant Amaranthus retroflexus seeds do not germinate in the dark at temperatures below 35°C. Fully dormant seeds germinate only at 35–40°C whereas non-dormant ones germinate within a wider range of temperatures (15 to 40°C). Germination of non-dormant seeds requires at least 10% oxygen, but the sensitivity of seeds to oxygen deprivation increases with increasing depth of dormancy. 10^–6 to 10^–4 M ethephon, 10^–3 M 1-aminocyclopropane 1-carboxylic acid (ACC) and 10^–3 M gibberellic acid (GA₃) break this dormancy. In the presence of 10^–3 M GA₃ dormant seeds are able to germinate in the same range of temperatures as non-dormant seeds. The stimulatory effect of GA₃ is less dependent on temperature than that of ethephon, while ACC stimulates germination only at relatively high temperatures (25–30°C). The results obtained are discussed in relation to the possible involvement of endogenous ethylene in the regulation of germination of A. retroflexus seeds.Abbreviations ACC 1-aminocyclopropane 1-carboxylic acid - GA₃ gibberellic acid - SD standard deviation     

Some effects of plant growth regulators on tissue cultures of the marine red alga Agardhiella subulata (Gigartinales,Rhodophyta)

We examined whether auxins and cytokinins, either singly or in combination, stimulate cell division in tissue cultures of a red seaweed. Our experimental model consisted of filamentous and callus-like growths that developed from cross-sectional discs cut from young branches of Agardhiella subulata. Plant growth regulators were added to the medium to give combinations of an auxin with a cytokinin over a range of concentrations (1 µg L^–1 –10 mg L^–1). Several mixtures of auxins and cytokinins, as well as some single auxins, cytokinins and phenolics, stimulated cell division and growth in the tissue cultures beyond that of controls. The treatments that were effective included: phenylacetic acid/zeatin; phenylacetic acid/6-benzylaminopurine; -naphthaleneacetic acid/zeatin; 2,4,5-trichlorophenoxyacetic acid/6-benzylaminopurine; and indoleacetic acid/kinetin. High concentrations of cytokinins (i.e. 10 mg L^–1) inhibited the regeneration of plants in some of the cell cultures. These results provide further evidence that growth regulators can be used for the tissue culture of seaweeds and for the study of developmental phenomena in these plants.     

Effect of exogenous cytokinins on growth and somatic embryogenesis in anise cells (Pimpinella anisum L.)

A cell culture of anise was grown in the presence or absence of 2,4-dichlorophenoxyacetic acid (2,4-D). Application of isopentenyladenine or isopentenyladenosine (4·10^-8 to 4·10^-7 M) to the proembryonic culture (+2,4-D) yielded an increase of the cell density, in contrast to a proembryonic culture grown without exogenous application of cytokinins. Embryogenesis was induced by transferring the cells to a hormone-free medium. Embryo development was promoted by isopentenyladenine and isopentenyladenosine (5·10^-8 to 5·10^-7 M), higher concentrations (5·10^-6 M) inhibited embryogenesis. The effect of cytokinins on embryogenesis was only promotive until the third day of culture, i.e. coincident with cell growth rather than differentiation.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - i6Ade isopentenyladenine - i6Ado isopentenyladenosine     

Stimulatory effect of genistein and daidzein on protein synthesis in osteoblastic MC3T3-E1 cells: Activation of aminoacyl-tRNA synthetase

The effect of genistein and daidzein on protein synthesis in osteoblastic MC3T3-E1 cells in vitro was investigated to determine a cellular mechanism by which the isoflavones stimulate bone formation. Cells were cultured for 48 h in -minimal essential medium containing either vehicle, genistein (10^–7–10^–5 M) or daidzein (10^–7–10^–5 M). The 5,500 g supernatant of cell homogenate was used for assay of protein synthesis with [³H]leucine incorporation in vitro. The culture with genistein or daidzein caused a significant elevation of protein synthesis in the cell homogenate. The effect of genistein (10^–5 M) or daidzein (10^–5 M) in elevating protein synthesis was significantly prevented, when cells were cultured for 48 h in a medium containing either actinomycin D (10^–7 M) or cycloheximide (10^–6 M) in the absence or presence of isoflavones. Moreover, when genistein (10^–7–10^–5 M) or daidzein (10^–6 and 10^–5 M) was added to the reaction mixture containing the cell homogenate obtained from osteoblastic cells cultured without isoflavone, protein synthesis was significantly raised. This increase was markedly blocked by the addition of cycloheximide (10^–7 M). In addition, [³H]leucyl-tRNA synthetase activity in the cytosol of osteoblastic cells was significantly increased by the addition of genistein (10^–6 and 10^–5 M) or daidzein (10^–5 M) into the enzyme reaction mixture. The present study demonstrates that genistein or daidzein can stimulate protein synthesis in osteoblastic MC3T3-E1 cells. The isoflavones may have a stimulatory effect on osteoblastic bone formation due to increasing protein synthesis.     

Phonolic components of the primary cell wall and their possible rôle in the hormonal regulation of growth

The insoluble cell wall polymers of cultured spinach cells contained esterified ferulic acid at 2–5 mg g^-1 dry weight. Gibberellic acid (GA₃, 10^-11–10^-6 M) promoted the expansion of these cells and simultaneoulsy suppressed peroxidase secretion, reduced the activity of cellular phenylanine ammonia-lyase and favoured the accumulation of wall-esterified ferulate and of extracellular soluble phenolic aglycones. When growth was prevented with 0·7 M sorbitol, GA₃ still evoked the phenolic and peroxidase effects. It is suggested that peroxidase restricts growth by rigidifying the cell wall in two ways: (a) covalently by catalysing the conversion of feruloyl side-chains into diferuloyl cross-links and (b) non-covalently by catalysing the conversion of soluble phenolics into hydrophobic quinones (or polymers). GA₃ is hypothesised to prevent this rigidification by inhibiting peroxidase secretion.Abbreviations A ₂₈ absorbance at 280 nm - a _1cnt ^1% absorptivity coefficient - 2,4-D 2,4-dichlorophenoxyacetic acid - EtOAc ethylacetate - GA₃ gibberellic acid - mol wt molecular weight - PAL phenylalanine ammonia-lyase - PCV packed cell volume - sh shoulder or inflection - TLC thin-layer chromatography - UV ultra-violet - wavelength - IAA indoleacetic acid