Hormonal Control of Cell Proliferation and Xylem Differentiation in Cultured Tissues of Glycine max var. Biloxi

The relationship between tracheary element differentiation, cell proliferation and growth hormones was examined in agar-grown soybean callus. The time course of cell division and tracheary element formation in tissues grown on a medium containing 5 x 10(-7)m kinetin and 10(-5)m NAA was determined by means of maceration technique. After a slight lag period, a logarithmic increase in cell number was observed through the twelfth day of the culture period. Cell numbers increased at a considerably slower rate after the twelfth day. The rate of tracheary element formation varied with the rate of cell proliferation. Tracheary elements increased logarithmically during the log phase of growth. As the rate of cell division decreased after the twelfth day of culture, the rate of tracheary element formation also decreased. In the presence of 10(-5)m NAA, cell number increased as the kinetin concentration was increased between 10(-9) and 10(-6)m. However, tracheary element formation was not initiated unless the kinetin concentration was 5 x 10(-8)m or above. When the Biloxi callus was subcultured repeatedly on media containing 10(-8)m kinetin, a tracheary element-free population of cells was obtained. This undifferentiated tissue produced tracheary elements upon transfer to a medium containing 5 x 10(-7)m kinetin. In the presence of 5 x 10(-7)m kinetin, NAA stimulated cell proliferation between 10(-7) and 10(-5)m, but no tracheary elements were formed without auxin, or with 10(-7)m NAA. Neither NAA nor kinetin at any concentration tested stimulated tracheary element formation in the absence of an effective level of the other hormone. However, 2,4-D at 10(-7) or 10(-6)m promoted both cell proliferation and tracheary element differentiation in the absence of an exogenous cytokinin.     

The influence of kinetin on the endogenous content of indoleacetic acid in swelling seeds of Phaseolus, Zea and Pinus and young plants of Phaseolus

Treatment of different plant materials, seeds of Phaseolus vulgaris, Zea mays and Pinus silvestris and young plants of Phaseolus, with kinetin increased the level of extractable IAA. For seeds this increase was most pronounced in bean seeds, which contained the lowest amount of endogenous IAA and cytokinins, and lower in maize seeds with high endogenous content of IAA and cytokinins. – For young bean plants the kinetin treatment significantly increased the extractable amounts of IAA from all parts of the plant, hypocotyls, cotyledons, epicotyls and primary leaves, when the cut plants were placed for 24 h in kinetin solution. For plants sprayed with kinetin solution only the primary leaves showed a significantly higher level of extractable IAA, which could be explained by the fact that the plants were growing very close together, so that the primary leaves received most of the kinetin during spraying.     

Organogenesis and somatic embryogenesis in Foeniculum vulgare: histological observations of developing embryogenic callus

Different NAA plus kinetin or BA combinations were tested on Francia Pernod fennel seedlings for callus induction and plant regeneration. Callogenesis from hypocotyls was obtained in all auxin/cytokinin-containing media. The organogenic response was observed especially in presence of NAA plus kinetin. The highest frequency of shoot regeneration was found when the auxin and kinetin were used at a 1:1 ratio. Moreover, a prolonged culture period increased shoot formation. Somatic embryogenesis was tested on several fennel populations. The results gave evidence of the genotypic importance. Two different protocols were used for somatic embryo induction. Using the first protocol among the different fennel genotypes tested, only Francia Pernod showed embryogenic capacity. In this case, from a primary non-embryogenic callus cultured for 12 months in presence of 2,4-D, an embryogenic secondary callus was produced. When transferred to the medium without 2,4-D (agarized or liquid), this gave embryogenic plants in high frequency. As far as the second embryogenic method is concerned, secondary embryogenic callus developed only in the presence of 2,4-D plus kinetin in Francia Pernod genotype. Thereafter, the replacement of those growth regulators by GA₃ into the medium greatly increased the somatic embryo development, especially in `Francia Pernod', but also in `Aboca erbe' callus, a population with a very poor embryogenic capacity. In Francia Pernod, the primary and secondary (embryogenic) calli showed different morphological and histological responses, either when the secondary callus was induced by 2,4-D alone or by 2,4-D plus kinetin. Ontogenetic processes leading to somatic embryo formation are described in this context. This revised version was published online in June 2006 with corrections to the Cover Date.     

Regulation of lateral root formation in lettuce (Lactuca sativa) seedling roots: Interacting effects of α-naphthaleneacetic acid and kinetin

Growth substances, α-naphthaleneacetic (NAA) and kinetin, had an important role in the regulation of lateral root (LR) formation in lettuce ( Lactuca sativa L. cv. Grand Rapids) seedling roots. NAA (10^-5 M ) was a potent stimulator of LR initiation and caused a 600% increase in the number of lateral root primordia (LRP) compared to untreated roots. NAA was required for only the first 20 h of the 72 h treatment period for maximum stimulation of LRP initiation. Kinetin (2 × 10^-5 M ) effectively prevented the spontaneous formation of LRP and inhibited the NAA-stimulated production of LRP. Kinetin inhibition was maximal during the first 20 h of NAA treatment and this effect was not overcome by subsequent supply of NAA. Also, lettuce roots were most sensitive to kinetin at 20 h of NAA treatment, when the first signs of cell division were observed in the pericycle.     

Somatic embryogenesis in the opium poppy, Papaver somniferum

A procedure is described for the induction of somatic embryos in the opium poppy. Papaver somniferum L. Callus was obtained from seedling hypocotyls on an agar solidified medium [Murashige and Skoog (1962) Physiol. Plant. 15: 473–497] containing 0.25 mg/l (1.2 μ M ) kinetin and 2.0 mg/l (10.7 μ M ) naphtalene acetic acid (NAA). Suspension cultures were initiated from callus using a liquid medium in which 2.0 mg/l (9.0 μ M ) 2,4-dichlorophenoxyacetic acid was substituted for NAA. Meristemoids, spheres of closely packed cells, developed in suspensions and on the surface of a few callus cultures. Differentiation of meristemoids into somatic embryos was accomplished by removing growth regulators from the liquid medium. Embryoids appeared morphologically normal and similar to torpedo stage embryos, however, they possessed mature tracheary elements and laticifers in areas that should have contained only procambium. Whole plants have been obtained by placing embryos in the light on solid medium that also lacked growth regulators.     

Kinetin and naphtaleneacetic acid controlled starch formation in isolated roots ofPisunt sativum

The formation of starch in excised 3-days-old pea roots was enhanced up to three fold after cultivation for five days in liquid media containing 3% glucose, microelements, and 10^-6 M kinetin or 10^-7 M naphtaleneacetic acid (NAA). A synergistic effect of kinetin and NAA on starch formation was observed when both hormones were applied simultaneously over a wide range of concentrations.     

Morphogenic capability of <Emphasis Type="Italic">Gentiana kurroo</Emphasis> Royle seedling and leaf explants

Experiments have been carried out on seedling and primary leaf explants of Gentiana kurroo Royle. Morphogenic capacities of cotyledons, hypocotyls and roots were investigated using MS (1962) medium supplemented with 4.64 μM kinetin and 2.26, 4.52 or 9.04 μM 2,4-D. Percentage of callusing explants for each combination was inversely proportional to numbers of obtained embryos. Cotyledons showed the highest morphogenic capabilities. To assess the morphogenic potential of leaf explants, 189 combinations of auxin (NAA, dicamba and 2,4-D) and cytokinin (kinetin, BAP, zeatin, CPPU and TDZ) in different concentrations were tested. The presence of NAA with BAP and dicamba with zeatin produced the greatest number of differentiated somatic embryos. Microscopic analysis of responsive explants led to identifying rhizogenic centers, non-embryogenic and embryogenic cells. The best embryo conversion into germlings was obtained on MS medium containing 4.46 μM kinetin, 1.44 μM GA₃ and 2.68 μM NAA or ½ MS. Both media were supplemented with 4.0% sucrose and 8.0% agar. Depending on explant origin and conversion medium, 55.8–71.0% of somatic embryos developed into germlings and plants.     

Plant regeneration via somatic embryogenesis from protoplasts of six explants in Coker 201 (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>)

Fertile regenerated plants were obtained from protoplasts via somatic embryogenesis in Coker 201 (Gossypium hirsutum L.). Protoplasts were isolated from six different explantsleaves, hypocotyls, young roots, embryogenic callus, immature somatic embryos and suspension cultures and cultured in liquid thin layer KM8P medium. Callus-forming percentage of 20–50% was obtained in protoplast cultures from embryogenic callus, immature embryos and suspension cultures, and visible callus formed within 2 months. Callus-forming percentage of 5–20% in protoplast cultures from young roots, hypocotyls and leaves, and visible callus formed in 3 months. NAA 5.371 μM/kinetin 0.929 μM was effective to stimulate protoplast division and callus formation from six explants. Percentage of callus formation in the medium with 2,4-D 0.452 μM/kinetin 0.465 μM was over 40% from suspension cultures and immature embryos, 25% from embryogenic callus and 10% from hypocotyls. Callus from protoplasts developed into plantlets via somatic embryogenesis. Over 100 plantlets were obtained from protoplasts derived from 6 explants. Ten plants have been transferred to the soil, where they all have set seeds.     

Promotion of hypocotyl elongation in loblolly pine (Pinus taeda L.) by indole-3-acetic acid

Elongation of excised loblolly pine ( Pinus taeda L.) hypocotyls was promoted by indole-3-acetic acid and the fungal metabolite, fusicoccin. Gibberellic acid, kinetin, zeatin, or zeatin-riboside were either without effect or promoted elongation only slightly. The most auxin-responsive tissue was just below the cotyledonary node, and elongation was confined to sections excised from the upper 2 cm of the hypocotyl. Indole-3-acetic acid induced elongation rates in the hypocotyl sections equal to those of intact hypocotyls when the sections were excised from young seedlings. Elongation rates decreased in intact hypocotyls before the excised tissues lost responsiveness to the auxin. Hypocotyl elongation in loblolly pine is discussed in relation to hypocotyl elongation in angiosperm species.     

Somatic embryogenesis in liquid cultures of a tetraploidAlstroemeria

A simple and efficient procedure was developed for regeneration of a tetraploid cultivar ofAlstroemeria (A. pelegrina x A. psittacina) via somatic embryogenesis in liquid cultures. Embryogenic callus induced from mature zygotic embryos, cultured on MS medium supplemented with 40 μM NAA and 20 μM kinetin, was used as inoculum for liquid cultures. Pre-culture of the callus on MS medium supplemented with 80 μM NAA for two days was essential for cell proliferation in the liquid medium. Embryogenic cell aggregates, obtained by sieving through a 750 μm nylon mesh, continued to proliferate in media containing 10 or 20 μM NAA and 10 or 20 μM kinetin. When transferred to a semi-solid half strength MS medium supplemented with casein hydrolysate, cell aggregates successfully differentiated into plantlets which later grew to maturity under greenhouse conditions.     

Morphogenesis in leaf,hypocotyl and root explants of Digitalis thapsi L. cultured in vitro

The effects of the auxins 2,4-D, NAA and IAA either alone or in combination with kinetin or BA were investigated to assess the morphogenetic potential of leaf, root and hypocotyl explants of Digitalis thapsi. Calluses were obtained from the three explants in basal medium without the addition of growth regulators and in leaves, the calluses formed roots. Application of 2,4-D, NAA or BA increased callus formation. The presence of NAA induced root formation and that of BA induced shoot formation via callus interphase. Indole-3-acetic acid alone only induced the generation of roots in the hypocotyl callus. Kinetin was ineffective in all the explants tested. Combinations of NAA with kinetin or BA were more effective in inducing organogenesis in leaf explants. Optimum responses were obtained in hypocotyl and root explants by using IAA in combination with BA, the highest rate of shoot regeneration being observed in hypocotyl explants.Rooting of the differentiated shoots was readily achieved in media without growth regulators. Regenerated plantlets were transferred to soil and grew with a survival rate of 70%.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid, Kin-kinetin - NAA naphthaleneacetic acid     

The isolation, culture and division of protoplasts from citrus cotyledons

High yields (2.3 × 10⁵ to 1.3 × 10⁶ protoplasts/g.f.wt.) of isolated protoplasts were obtained from cotyledons of Cirus sinensis (L.) Osb. 'Valencia'. Osmotic potential of the medium and enzyme concentrations were important in obtaining high viability of preparations as indicated by FDA fluorescence. Adding malt extract to a Murashige-Tucker basal medium increased plating efficiencies somewhat, but not the rate or duration of cell division. However, modifying the NAA and kinetin concentration optimized plating efficiencies (up to 20%) of protoplasts and also the rate or duration of cell division. The highest plating efficiency and number of cells per colony were obtained on a defined medium containing NAA (15 μ M ). and kinetin (4.6 μ M ). Coincidence of percentage protoplast viability after 13 days (assessed by FDA fluorescence) with plating efficiency after 21 days indicates that FDA fluorescence is an accurate indicator of citrus protoplast viability.     

Effect of gibberellic acid, kinetin and naphthylacetic acid on the growth of coconut seedlings

The effects of three growth regulators, namely gibberellic acid (GA₃), kinetin (6-furfuryl-aminopurine) and α-naphthyl-acetic acid (NAA), each at five concentrations (0, 25, 50,10² and 10³ mg/litre) on the growth of coconut seedlings (Cocos nucifera) were examined. GA₃ at 25 mg/litre increased growth in height; higher concentrations had no significant effect. Kinetin at 10² and 10³ mg/litre and NAA at all concentrations reduced height growth compared with the control. No treatment caused a significant increase in total dry weight.     

High frequency plant regeneration from stem explants of Brassica alboglabra Bailey in vitro

Culture conditions for shoot regeneration and proliferation, and rooting of Brassica alboglabra Bailey were optimized by a judicious selection of explants and manipulation of hormonal combinations in the culture medium. Both half and whole stem explants were more regenerative than cotyledons and hypocotyls. The highest shoot regeneration frequency (100%) accompanied by high number of shoots was obtained using half stem explants grown on Murashige & Skoog [14] medium supplemented with 2 mgl^-1 BA in combination with 1 mgl^-1 NAA, or 4 mgl^-1 2iP with 0.5 mgl^-1 NAA. For shoot proliferation, 4 mgl^-1 kinetin was most effective. The presence of auxin reduced shoot proliferation significantly. Maximum rooting (100%) of shoot cuttings was obtained either in the absence or in the presence of 0.5 mgl^-1 NAA, or IBA or IAA ranging from 0.1 to 8 mgl^-1.     

The interaction of auxin and cytokinin in the induction of phenylalanine ammonia-lyase in suspension cultures of Phaseolus vulgaris

The dependence of phenylalanine ammonialyase (PAL) induction in bean suspension cultures on the concentration of naphthylacetic acid (NAA) and kinetin has been investigated and the timing of the effect of each hormone has been determined. NAA was required at an optimal concentration of 1 mg l^-1 2 days prior to the increase in PAL activity. Kinetin caused a prapid stimulation of the rate of PAL induction and the total amount of PAL induced in a concentration range of 0.1–0.5 mg l^-1 when it was supplied to the cells immediately prior to the expected rise in PAL activity. The inhibitory effect of 2 mg l^-1 NAA on PAL induction was overcome by an increased concentration of kinetin.Abbreviations NAA naphthylacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3yl acetic acid - PAL phenylalanine ammonia-lyase (EC 4.3.1.5.)     

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     

Opposite effects of synthetic auxins, 2,4-dichlorophenoxyacetic acid and 1-naphthalene acetic acid on growth of true ginseng cell culture and synthesis of ginsenosides

Effects of synthetic auxins (2,4-D and NAA) on growth of true ginseng (Panax ginseng C.A. Mey) suspension culture and ginsenoside synthesis were investigated. Cell suspensions were grown for 6–8 subcultures on media supplemented with various phytohormones. In all media supplemented with 2,4-D and cytokinins (benzyladenine or kinetin), the cell culture showed sustained growth both in the presence and absence of casein hydrolysate. The average growth index, determined from fresh weight increment over one subculture, equaled to 5.16 ± 0.90, and the maximum mitotic index was 2%. These cell populations having cell volume of 10–17 × 10⁴ μm³ were composed mostly (up to 60–80%) of 5-to 10-cell aggregates with unimodal distribution of nuclear DNA. These cell suspensions were suitable for isolation of protoplasts. The total average content of ginsenosides in the cell culture grown in the presence of 2,4-D constituted 0.18% of dry matter. In media supplemented with NAA, the cell growth was retarded irrespective of the cytokinin species and presence or absence of casein hydrolysate. The growth index (the ratio of final to initial fresh weights) was on average 2.15 ± 0.37, and the mitotic index did not exceed 0.13%. These suspensions, characterized by cell volume of 22–50 × 10⁴ μm³, were composed of large aggregates (> 50 cells). The attempts to isolate protoplasts from these suspensions were unsuccessful. About 25% of cells cultured in the presence of NAA had doubled nuclear DNA content by the end of the subculture. The total content of ginsenosides in cell cultures grown with NAA was on average 4.46% of cell dry matter. The results indicate that ginsenoside synthesis depends on the extent of differentiation in the population of true ginseng cells grown in suspension culture. A certain extent of cytodifferentiation in the cell culture was observed in the presence of NAA, whereas 2,4-D supported only cell proliferation in vitro.     

Growth Hormones and Propagation of Cymbidium in vitro

Protocorms of Cymbidium (Orchidaceae) were grown on solid or liquid medium with macro-nutrients according to Wimber (van Raalte 1967) and iron, micro-nutrients and vitamins according to Nitsch (1968) the medium also contained 2% sucrose. The effects of 1) the auxins; indol-3yl-acetic acid (IAA), α-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D); 2) the cytokinins; 6-furfurylaminopurine (kinetin) and benzyladenine (BA) and 3) the gibberellin; gibberellic acid (GA) were examined alone or in combinations. IAA had no effect alone. NAA resulted in optimal fresh weight at 10 μM and the protocorms were vigorous, but lighter green than usual. 2,4-D caused a high weight increase at 1 μM, but the protocorms were abnormal. Higher concentrations of NAA and 2,4-D inhibited chlorophyll synthesis. On solid medium kinetin (100 μM) induced a growth of many small shoots, but had no effect on the fresh weight. In liquid medium, kinetin promoted a callus formation and fresh weight increase. BA had effects similar to kinetin, but at lower concentrations. GA alone promoted shoot and leaf growth. Combinations of kinetin and NAA resulted in a maximal fresh weight increase at kinetin concentrations one tenth of the NAA concentrations. The optimal growth and the best development occurred at 10 μM NAA and 1 μM kinetin. NAA and kinetin together could limit the shoot and leaf growth induced by GA.     

The Effect of Plant Hormone on Callus Formation and Regeneration of Plant in Cucumis melo L. var. Outvmnesles fil

The calli could be induced from the cotyledon in Miller medium supplemented with high concentration of kinetin (5–10 ppm) or with 2 ppm NAA plus 0.5 ppm kinetin. The callus induced by NAA plus kinetin was much different from that by kinetin alone. The former was loose and soft, while the later was tight and firm. About half a month after inoculation the later formed globe-like tissues and then new buds initiated and developed. The roots initiated from the buds when it was transplanted into Miller medium with low concentration of kinetin. The plantlets were thus obtained. However, the plantlet could not be gotten in white medium or Miller medium when hypocotyl explants were used. Therefore, it is concluded that regulation of callus formation and differentiation of the calli by exogenous hormone is closely related to some endogenous factors and external condition.