In vitro PPFD and media composition affect both in and ex vitro performance of Alstroemeria Butterfly-hybrids

The objective was to reduce in vitro production costs while retaining or improving plant quality, in particular the suitability for pot plant production. Plants were grown at photosynthetic photon flux densities (PPFD) of 0–40 μmol m^-2 s^-1 and sucrose concentrations of 3–7% during the multiplication phase and the effects of sucrose, BA, and NAA during root formation were investigated. Ex vitro growth were tested in both experiments. A small reduction in the rhizome multiplication rate was found with increasing PPFD and sucrose concentration. Increasing sucrose concentration reduced the number of aerial shoots. Aerial shoots were etiolated when cultured in darkness and their number increased with increasing PPFD at 3% sucrose, whereas PPFD did not affect the number of aerial shoots at 5 or 7% sucrose. During the multiplication phase a synergistic promoting effect of PPFD and sucrose was observed on root formation. Root formation after transfer to rooting medium was affected by sucrose and PPFD during the multiplication phase. PPFD did not influence root formation after propagation on 7% sucrose, whereas on 3 or 5 % sucrose root formation was gradually inhibited when PPFD was decreased below 17 μmol m^-2 s^-1. The formation of thick roots was promoted by propagation in light, and not influenced by sucrose concentration. Ex vitro growth was not affected by in vitro conditions, except for 7% sucrose during the multiplication phase that reduced flowering. Root formation on rooting medium was reduced by BA and promoted both by NAA and high levels of sucrose. The root inhibiting effect of BA could not completely be overcome by simultaneous application of NAA and high sucrose concentrations. Thick roots were only produced in the presence of NAA, and not affected by sucrose treatment. Ex vitro flowering was negatively influenced by the presence of BA during root formation and by high levels of sucrose if BA was absent in the rooting medium. High sucrose levels and NAA could partially compensate for the negative effect of BA on flowering. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro shoot organogenesis from excised immature cotyledons and microcuttings production in Stone Pine

Adventitious buds were induced on isolated immature cotyledons of Pinus pinea L. in the presence of benzyladenine (BA). The response to different BA concentrations also depended upon the culture medium used (modified MS, SH and GD). A wide range of BA concentrations (5, 25 or 50 M) can be applied to the GD and SH media, which are the media with the lower nitrogen content, without damaging effects. In the MS medium, which has the highest nitrogen concentration, the range of BA that can be applied was narrower and the highest BA concentration was lethal. The addition of indolebutyric acid (0.05, 0.25 or 0.5 M) to the induction medium, decreased the response of cotyledons. The increase in the concentration of sucrose from 3% to 5% did not increase the number of responding cotyledons. The addition of activated charcoal (0.5 and 3 g l^-1) or indolebutyric acid (1.5 or 3 M) did not speed up the elongation of explants. Elongation of the buds produced shoots with two different phenotypes, each phenotype having a different multiplication rate.Abbreviations BA benzyladenine - GD Gresshoff & Doy medium - IBA indolebutyric acid - MS Murashige & Skoog medium - SH Schenk & Hildebrandt medium     

Micropropagation of mature <Emphasis Type="Italic">Quercus ilex</Emphasis> L. trees by axillary budding

This paper reports the successful micropropagation of mature Quercus ilex trees known as reluctant to in vitro propagation. Crown branch segments collected from 30 to 100 year-old trees were forced in order to promote the production of sprouting shoots that were used as a source of explants for initiating the cultures. Sterilization was critical and required low-level disinfestation protocols. Six out of the eight mature genotypes attempted were successfully inoculated and then maintained in culture with varying responses. Shoot proliferation of holm oak was influenced by BA concentration, with improved multiplication and shoot appearance when the BA concentration was sequentially reduced over the culture period. Micropropagation by axillary budding was achieved by culturing shoots on a sequence of cytokinin-enriched Lloyd and McCown (WPM) media alternating 2 week-long subcultures on 0.44 µM benzyadenine (BA) first, followed by 0.22 µM BA, then 0.044 µM BA plus 0.46 µM zeatin. Sucrose concentration and agar brand affected shoot proliferation, and the best results were obtained on WPM medium supplemented with 8 g L^?1 Sigma agar (A-1296; Sigma-Aldrich) and 30 g L^?1 sucrose. Addition of 20 µM silver thiosulphate had a significant positive effect on the appearance and development of shoots with a higher number of shoots being healthy and showing reduced shoot tip necrosis and early senescence of leaves. The 18.8% of the microshoots obtained for one clone could be rooted within 15 days on a half-strength Murashige and Skoog medium containing 14.8 µM or 24.6 µM indole-3-butyric acid and 0.54 µM α-naphthalene acetic acid.     

PROMOTION OF PLASTID PIGMENT ACCUMULATION IN WATER STRESSED WHEAT LEAF SECTIONS BY HORMONE TREATMENT

Chlorophyll (Chl) and carotenoid accumulations are markedly reduced in detached wheat leaves after 20-hr exposure to light when subjected to mild (-7 bar) osmotic stress prior to illumination. The treatment of N⁶-benzyladenine (BA) in combination with gibberellic acid (GA) stimulated the recovery of plastid pigment accumulations in mildy stressed detached leaves. The combination treatment of BA plus IAA increased pigment accumulations only to the level stimulated by BA treatment alone. Mild water deficits increased the Chl a/b ratio and reduced the Chl/carotenoid ratio in wheat sections. Growth promoting hormones generally lowered the Chl a/b ratio and increased the Chl/carotenoid ratio in mildly stressed detached leaves. Abscisic acid (ABA) reduced plastid pigment concentrations in greening tissue and increased the Chl a/b ratio while decreasing the Chl/carotenoid ratio. In detached leaves stressed to -14 bars, BA in combination with GA stimulated only partial recovery of the plastid pigment accumulations and improved slightly the Chl a/b and Chl/carotenoid ratios.     

Somatic embryogenesis and plant regeneration of Nerium oleander

Leaf explants of Nerium oleander L. produced masses of callus when both an auxin and a cytokinin were included in the medium. Leaves cultured on the B5 medium of Gamborg et al. supplemented with 2,4-dichlorophenoxyacetic acid (2,4-d; 9.05 M) plus benzyladenine (BA; 4.4 M) produced callus and profuse rhizogenesis was observed from callus developed from older leaves. On Murashige & Skoog medium (MS) with the same concentration of 2,4-d and BA, explants from young and mature leaves produced callus, but only that from young leaves was embryogenically competent. Globular somatic embryos were obtained when embryogenic cells were cultured on MS medium without growth regulators. Both normal and anomalous development of embryos occurred in either liquid or gelled medium. Plantlets were produced faster when mature embryos were cultured on either solid medium or placed on Sorbarod plugs soaked with this same medium but with 1% sucrose. Plantlets with three nodes were transferred to pots and acclimatized in a growth chamber and afterwards transferred to garden beds.     

Embryogenesis and plant regeneration from unpollinated ovary culture of <Emphasis Type="Italic">Psoralea corylifolia</Emphasis>

Embryogenesis and plant regeneration was achieved from callus cultures derived from unpollinated ovaries of Psoralea corylifolia L. Callus was initiated from unpollinated ovaries on Murashige and Skoog (MS) medium supplemented with 2.2 μM N ⁶-benzyladenine (BA) and various concentrations of α-naphthaleneacetic acid (NAA (2.7 to 10.7 μM) or 2,4-dichlorophenoxyacetic acid (2,4-D (2.3 to 9 μM) alone or in combination. Highly organized embryogenic callus induction, embryo development, proliferation and maturation were achieved on transfer of callus clumps to MS medium supplemented with NAA (0.27 μM) or 2,4-D (0.23 μM) alone or in combination with BA (2.2 to 8.8 μM). Addition of abscisic acid (ABA) (0.95 to 5.8 μM) to the medium enhanced average numbers of cotyledonary stage embryos, the maximum number (34.6 ± 0.7) being obtained on MS medium containing 0.27 μM NAA, 2.2 μM BA and 3.8 μM ABA. Embryos germinated on MS medium supplemented with BA (0 to 8.8 μM). MS medium containing gibberellic acid (GA₃ (0.29 to 5.8 μM) enhanced embryo germination frequency, the highest frequency (66.7 %) occurring on MS medium containing 2.2 μM BA and 4.3 μM GA₃. Effect of several concentrations (3.0 to 6.0 %) of sucrose or maltose was also observed on germination of embryos. MS medium enriched with maltose supported high frequency of embryo germination.     

Regeneration of zoysia grass (Zoysia matrella L. Merr.) cv. Konhee from young inflorescences and stem nodes

We have optimized conditions for efficient regeneration of the vegetatively propagated zoysia grass (Zoysia matrella L. Merr) cultivar “Konhee”. Two explants, young inflorescences, and stem nodes, were used and they displayed different responses to combinations and concentrations of plant growth regulators in callusing, embryogenic callus formation, and regeneration. The highest callus initiation rate from young inflorescences was obtained on medium supplemented with 4.5 to 9.0 μM 2,4-dicholorophenoxy acetic acid (2,4-D) and 0.44 μM 6-benzyl amino purine (BA). When the BA concentration was lowered to 0.044 μM, the highest percent embryogenic callus induction from young inflorescences was achieved. The highest callus initiation rate from stem nodes was obtained, when young inflorescences were cultured on MS medium supplemented with 4.5 to 9.0 μM 2,4-D, 0.44 μM BA, and 0.037 μM abscisic acid (ABA). But embryogenic callus formation from the stem node was highest in the presence of 4.5 to 9.0 μM 2,4-D, 0.044 μM BA, and 0.037 μM ABA. Addition of ABA significantly increased embryogenic callus formation from stem nodes, but not from young inflorescences. Regeneration percentage was variable in response to BA level, and inclusion of α-naphthalene acetic acid (NAA) and gibberellic acid (GA₃) further increased the regeneration percentage. The highest regeneration percentages obtained from the young inflorescences and stem nodes were 82% and 67%, respectively. This is the first report showing that plants can be regenerated from young inflorescences and stem nodes of vegetatively propagated zoysia grass.     

Physicochemical and biological properties of natural and synthetic C-22 and C-23 hydroxylated bile acids

In order to define the effect of a side chain hydroxy group on bile acid (BA) physicochemical and biological properties, 23-hydroxylated bile acids were synthesized following a new efficient route involving the alpha-oxygenation of silylalkenes. 22-Hydroxylated bile acids were also studied. The synthesized bile acids included R and S epimers of 3 alpha,7 alpha,23-trihydroxy-5 beta-cholan-24-oic acid (23R epimer: phocaecholic acid), 3 alpha,12 alpha,23-trihydroxy-5 beta-cholan-24-oic (23R epimer: bitocholic acid), and 3 alpha,7 beta,23-trihydroxy-5 beta-cholan-24-oic acid. A 3 alpha,7 alpha,22-trihydroxy-5 beta-cholan-24-oic acid (haemulcholic acid) was also studied. The presence of a hydroxy group on the side chain slightly modified the physicochemical behavior in aqueous solution with respect to common BA: the critical micellar concentration (CMC) and the hydrophilicity were similar to naturally occurring trihydroxy BA such as cholic acid. The pKa value was lowered by 1.5 units with respect to common BA, being 3.8 for all the C-23 hydroxy BA. C-22 had a higher pKa (4.2) as a result of the increased distance of the hydroxy group from the carboxy group. When the C-23 hydroxylated BA were intravenously administered to bile fistula rats, they were efficiently recovered in bile (more than 80% unmodified) while the corresponding analogs, lacking the 23- hydroxy group, were almost completely glycine- or taurine-conjugated. On the other hand, the C-22 hydroxylated BA were extensively conjugated with taurine and less than 40% of the administered dose was secreted without being conjugated. In the presence of intestinal bacteria, they were mostly metabolized to the corresponding 7-dehydroxylated compound similar to common BA with the exception of bitocholic acid which was relatively stable. The presence of a hydroxy group at the C-23 position increased the acidity of the BA and this accounted for poor absorption within the biliary tree and efficient biliary secretion without the need for conjugation. 3 alpha,7 beta-23 R/S trihydroxy-5 beta-cholan-24-oic acids could improve the efficiency of ursodeoxycholic acid (UDCA) for gallstone dissolution or cholestatic syndrome therapy, as it is relatively hydrophilic and efficiently secreted into bile without altering the glycine and taurine hepatic pool.     

Components of auxin transport in stem segments of Pisum sativum L.

1. The uptake of indol-3-yl acetic acid ([1-¹⁴C]IAA, 0–2.0 M) into light-grown pea stem segments was measured under various conditions to investigate the extent to which mechanisms of auxin transport in crown gall suspension culture cells (Rubery and Sheldrake, Planta 118, 101–121, 1974) are also found in a tissue capable of polar auxin transport. — 2. IAA uptake increased as the external pH was lowered. IAA uptake was less than that of benzoic acid (BA), naphthylacetic acid (NAA) or 2,4 dichlorophenoxyacetic acid (2,4D) under equivalent conditions. TIBA enhanced net IAA uptake through inhibition of efflux, and to a lesser extent, also increased uptake of NAA and 2,4D while it had no effect on BA uptake. — 3. Both DNP and, at higher concentrations, BA, reduced IAA uptake probably because of a reduction of cytoplasmic pH. However, low concentrations of both BA and DNP caused a slight enhancement of IAA net uptake, possibly through a reduction of carrier-mediated IAA efflux. In the presence of TIBA, the inhibitory effects of DNP and BA were more severe and there was no enhancement of uptake at low concentrations. — 4. Non-radioactive IAA (10 M) reduced uptake of labelled IAA but further increases in concentration up to 1.0 mM produced first an inhibition (0–10 min) of labelled IAA uptake, followed by a stimulation at later times. Non-radioactive 2,4 D decreased, but was not observed to stimulate, uptake of labelled IAA. In the presence of TIBA labelled IAA uptake was inhibited by non-radioactive IAA regardless of its concentration. — 5. Sulphydryl reagents PCMB and PCMBS promoted or inhibited IAA uptake depending, respectively, on whether they penetrated or were excluded from the cells. The penetrant PCMB also reduced the promotion of labelled IAA uptake by TIBA or by high concentrations of added non-labelled IAA. — 6. Our findings are interpreted as being consistent with the diffusive entry of unionised IAA into cells together with some carrier-mediated uptake. Auxin efflux from the cells also appears to have a carrier-mediated contribution, at least part of which is inhibited by TIBA, and which has a capacity at least as great as that of the uptake carrier. The data indicate that pea stem segments contain cells whose mechanisms of trans-membrane auxin transport fit the model of polar auxin transport proposed from experiments with crown gall suspension cells, although differences, particularly of carrier specificity, are apparent between the two systems.Abbreviations IAA indol-3-yl acetic acid - BA benzoic acid - NAA 1-naphthylacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - TIBA 2,3,5-triiodobenzoic acid - DNP 2,4-dinitrophenol - PCMB p-chloromercuribenzoic acid - PCMBS p-chloromercuribenzene sulphonic acid This work was performed in Cambridge during the tenure of a sabbatical leave by P.J.D. Supported by a grant for supplies from the American Philosophical Society to P.J.D.     

Morphogenic potential of mechanically isolated single cells from Digitalis obscura L. callus

Calli from hypocotyl and root explants of Digitalis obscura L. showed regeneration of adventitious shoots, roots and embryos when transferred to Murashige & Skoog medium supplemented with cytokinins alone or in combination with auxins. Optimum shoot-bud formation was achieved in the presence of IAA and BA, while roots mainly appeared either in absence of growth regulators or with IAA and Kn. Embryo formation took place only in those combinations that included Kn. Embryo development was influenced by the type of auxin, and precocious germination occurred in media with NAA. Mechanically isolated cells from hypocotyl- and root-derived calli were plated in MS medium supplemented with several IAA and BA combinations. Single cells were able to proliferate forming callus within 20–30 days in culture. In order to induce organogenesis, calli were transferred to various regeneration media. Shoot-bud differentiation efficiency depended on both callus origin and medium initially used for cell culture, best results being obtained in calli grown from hypocotyl-derived cells cultured in the presence of casein hydrolysate. A further subculture to medium containing coconut milk and lower concentrations of NH₄NO₃ and sucrose promoted shoot development. Rooting was readily achieved upon transferring shoots onto half-strength MS medium. Plantlets were ultimately established in soil.Abbreviations BA benzyladenine - BM basal medium - CH casein hydrolysate - CM coconut milk - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - Kn kinetin - MS Murashige & Skoog - NAA naphthaleneacetic acid     

In vitro studies on apogamy, apospory and controlled differentiation of rhizome segments of the fern, Ampelopteris prolifera (Retz.) Copel.

Apogamy was induced in the fern Ampelopteris prolifera by culturing the gametophytes on mineral nutrients supplemented with various concentrations of sucrose. Higher concentrations (5–8%) of sucrose were detrimental to prothallial growth, while in lower concentrations (2–3 %) apogamy was delayed. Gametophytic callus was induced from the germinating spores by culturing them on 2,4-D rich (3–5 mg/1) media. The differentiation of this gametophytic callus was conditioned by sucrose and auxin concentrations of the medium. In the presence of sucrose, calli responded like prothalli, while in the presence of 2,4-D, differentiation was delayed or completely inhibited. Apospory was induced on the sexual cotyledonary and juvenile sporeling leaveS. Leaves with petiole, excised from aseptically raised plants from excised cultured buds, also exhibited apospory, while no success was achieved with the excised leaves of the parent plantS. Rhizome segments of various length were cultured on media containing different concentrations of sucrose. The differentiation of rhizome segments into gametophytes or sporophytes was conditioned by the length of the rhizome segments and the sucrose concentration of the medium. The possible significance of all the results is discussed.     

Morphological and physiological responses of proliferating shoots of teak to temporary immersion and BA treatments

Shoot-tips, collected from greenhouse-grown plants of Tectona grandis L. (teak), were incubated on a semi-solid Murashige and Skoog (MS) medium with 2% (w/v) sucrose, and supplemented with 4.44 μM 6-benzyladenine (BA). These were then transferred to a temporary immersion system (TIS) using liquid MS medium supplemented with 0 (CK-free medium), 2.22, 4.44, 6.66 μM BA. High mean numbers of shoots per explant were obtained when explants were grown on medium containing either 4.44 or 6.66 μM BA and yielding 7.7 and 10.3 normal shoots (NS)/explant, respectively. Moreover, these high BA levels contributed to lower accumulation of phenolic compounds and deposition of lignin in vascular cells of the teak shoots following histochemical analysis. Morphological analysis of proliferating shoots by scanning microscopy revealed that leaves of shoots incubated on either CK-free medium, 2.22, or 4.44 μM BA had elliptical stomata; whereas, stomata of leaves of shoots grown on medium containing 6.66 μM BA were primarily ring-shaped, raised, and open. Moreover, misshapen stomata with broken epidermal layers of guard cells, typical of hyperhydric leaves, were also observed. When shoots were rooted ex vitro by dipping in 492.1 μM indole-3-butyric acid (IBA) for 2 min, the frequency of rooting of shoots previously grown on either CK-free medium or 2.22 μM BA (96.7 and 91.7%, respectively) was higher than that of shoots grown on semi-solid medium (73%). Shoots from both TIS treatments developed good root systems, and all plantlets (100%) survived transfer to soil mix and acclimatization in the greenhouse. Plantlets established from shoots grown on 6.66 μM BA showed the lowest frequency of survival (60%). After 3 months, plants were transferred to field conditions.     

Effects of sucrose concentration and exogenous hormones on growth and periplocin accumulation in adventitious roots of Periploca sepium Bunge

Periploca sepium adventitious roots were cultured on 0.5 Murashige and Skoog solid media supplemented with exogenous hormones of different types and various concentrations, and with sucrose of different concentrations. Auxins (indole butyric acid (IBA) and naphthalene acetic acid (NAA)) and cytokinins (6-benzylaminopurine (BA) and kinetin (KT)) were selected as exogenous hormones for adventitious root proliferation. Compared with other hormones, IBA was the suitable auxin for adventitious root proliferation. Under this circumstance, every root explant generates 10?C15 adventitious roots (1- to 2-cm long) after 30?days. However, nothing but callus was induced on the root explants when NAA was added into the medium and the same result was achieved when auxins (IBA or NAA) were added into the media together with cytokinins (BA or KT). The suitable concentration of IBA for adventitious root proliferation was 1?C2?mg/l, when every root explant generated 10?C20 adventitious roots (1- to 2-cm long). The optimum concentration of IBA for periplocin accumulation was 1?mg/l, when the periplocin content reached 95.46???g/g. With regard to the investigation of sucrose concentration, 2?C3% (w/v) sucrose was favorable for adventitious root proliferation as every root explant in this concentration generated 10?C20 adventitious roots (1- to 2-cm long). The highest periplocin content (101.56???g/g) was achieved at 5% (w/v) sucrose, whereas the periplocin content at 5% (w/v) sucrose did not show significant difference from the periplocin content (95.38 and 98.47???g/g, respectively) at 3% (w/v) or 4% (w/v) sucrose.     

Interaction of Growth Retardants and Temperature in Growth, Flowering, Regeneration, and Auxin Activity of Begonia × cheimantha Everett

Soil application of CCC reduced stem and leaf growth in Begonia plants. This effect was evident with all concentrations tested at 18°C, whereas at 21 and 24°C no growth–retarding effect was observed with 2 × 10^?2 M CCC, and with 5 × 10^?3 M growth was even stimulated. Flowering was promoted by CCC in long day and neur–critical temperature, particularly under low light intensity in the winter. The formation of adventitious buds in leaves of plants grown at 21 and 24°C was stimulated when the plants received 5 × 10^?2 and 2 × 10^?2 M CCC, while 8 7times; 10^?2 M was inhibitory. In plants grown at 18°C bud formation was inhibited by all CCC concentrations. Root formation in the the leaves was usually stimulated by high CCC concentrations, while root elongation was reduced. The level of ether–extractable. acidic auxin (presumably IAA) in the leaves was lowered by CCC treatment of the plants, hut this required higher CCC concentrations at higt than at low temperature. When applied to detached leaves CCC stimulated bud formation at concentrations ranging from 10^?4 to 10^?2 M in leaves planted at 18 and 21°C. At 24°C budding was inhibited by 10^?2 M CCC, the lower concentrations being stimulatory also at this temperature. Root formation and growth were not much affected by CCC treatment of the leaves, but increased with the temperature. Soil application of Phosfon (4 × 10^?4 M) had no effect on growth and flowering, nov did it affect the subsequent regeneration of buds and roots in the leaves. In detached leaves Phosfon stimulated bud formation with au optimum at 10^?6 M. Root formation was stimulated by Phosfon at all temperatures, the optimal concentration being 10^?5 M, whereas root length was conversely affected. Foliar application of B-995 to intact plants and treatment of detached leaves greatly inhibited the formation of buds and had little effect on root formation. B-99D reduced the growth and delayed flowering in the plants.     

Regulation of sucrose storage by amino acid arginine in sugarcane cell suspension cultures

Sugarcane cell cultures were obtained from callus formed on explants derived from young expanding leaves of two early maturing sugarcane varieties viz “CoJ83” and “CoJ86”. The cell cultures were varied with different arginine concentrations in the culture medium. For each cultivar, sucrose content with 20 μM arginine in the culture medium decreased from 3 to 5 days and then increased to 10 days after subculturing. Higher concentration of arginine in the culture medium (60 μM) decreased the sucrose content at different days after subculturing and thus significantly stimulated sucrose mobilization. The activity of sucrose synthase and sucrose phosphate synthase reached maximum while the activity of acid and neutral invertase was minimal in the culture medium with 20 μM arginine. Thus arginine at low concentration (20 μM) enables the cells to accumulate the higher level of sucrose. The optimum level of amino acids can be utilized to regulate the in vivo activity of sucrose synthase, sucrose phosphate synthase and invertase to achieve maximum sucrose accumulation in sugarcane storage tissue.     

Influence of benzyladenine and gibberellic acid on organogenesis in lsquo;Crimson Giantrsquo; Easter cactus

Experiments were performed to determine the influence of gibberellic acid (GA₃) and benzyladenine (BA) on organogenesis of lsquo;Crimson Giantrsquo; Easter cactus [Hatiora gaertneri (Regel) Barthlott] phylloclades cultured in vitro. The numbers of flower buds and new phylloclades increased linearly as BA concentration increased from 0 to 444.1 micro;M. GA₃ increased the number of new phylloclades when present in moderate concentrations (2.9 or 28.9 micro;M), but inhibited flower bud formation when present in concentrations as low as 0.3 micro;M. The inhibitory effect of GA₃ on flower bud formation was diminished when the medium was amended with BA at 44.4 or 444.1 micro;M. Explants cultured in media that contained 288.7 micro;M GA₃ produced fewer organs (new phylloclades plus flower buds) compared to those cultured in media with 0, 0.3, 2.9, or 28.9 micro;M GA₃. BA and GA₃ concentrations also affected the percentage of explants with flower buds and the percentage of explants with new phylloclades. This study shows that organogenesis in H. gaertneri can be controlled by varying the concentrations of BA and GA₃ in the culture medium.     

Improvement of somatic embryogenesis in zonal geranium

A number of media constituents including sucrose, ammonium nitrate and plant growth regulators were evaluated in an attempt to improve somatic embryo production in zonal geranium (Pelargonium ×hortorum) cv. Scarlet Orbit Improved. Somatic embryo production was characterized by the quantity and type of somatic embryo induced by the treatments. Sucrose at 4% supported the highest number of total somatic embryos while improving the proportion of the morphologically normal cotyledon-stage somatic embryos. Addition of ammonium nitrate also improved embryo production. With 1.89 mM ammonium nitrate, normal cotyledon-stage embryo development was increased by 53%; the proportion of normal cotyledon-stage embryos decreased and abnormal embryos with leaves or serrated margins in cotyledons (fringed-shoot type) increased with higher ammonium nitrate concentrations. The effect of plant growth regulators on somatic embryogenesis indicated that exogenous supply of indole-3-acetic acid (IAA) at a range of 0.25 to 4 μM failed to promote somatic embryogenesis. In contrast, benzyladenine (BA) up to 2.0 μM increased the total embryo number and the proportion of desirable cotyledon-stage embryos. There was no interaction between IAA and BA. Our research has demonstrated that improvement in both quantity and quality of somatic embryos can be achieved in zonal geranium.     

Improvement of somatic embryogenesis in zonal geranium

A number of media constituents including sucrose, ammonium nitrate and plant growth regulators were evaluated in an attempt to improve somatic embryo production in zonal geranium (Pelargonium ×hortorum) cv. Scarlet Orbit Improved. Somatic embryo production was characterized by the quantity and type of somatic embryo induced by the treatments. Sucrose at 4% supported the highest number of total somatic embryos while improving the proportion of the morphologically normal cotyledon-stage somatic embryos. Addition of ammonium nitrate also improved embryo production. With 1.89 mM ammonium nitrate, normal cotyledon-stage embryo development was increased by 53%; the proportion of normal cotyledon-stage embryos decreased and abnormal embryos with leaves or serrated margins in cotyledons (fringed-shoot type) increased with higher ammonium nitrate concentrations. The effect of plant growth regulators on somatic embryogenesis indicated that exogenous supply of indole-3-acetic acid (IAA) at a range of 0.25 to 4 µM failed to promote somatic embryogenesis. In contrast, benzyladenine (BA) up to 2.0 µM increased the total embryo number and the proportion of desirable cotyledon-stage embryos. There was no interaction between IAA and BA. Our research has demonstrated that improvement in both quantity and quality of somatic embryos can be achieved in zonal geranium.     

Senescence of rice leaves IV. Influence of benzyladenine on chlorophyll degradation

Why benzyladenine (BA) retards chlorophyll degradation of riceleaf segments, but does not prevent it was investigated. WhenBA solutions bathing segments of rice leaves were replaced daily,the effect of BA was not enhanced. Solutions of BA kept at 27°Cin the dark for 4 days were as active as fresh solutions. Itseems unlikely, therefore, that the decrease in the chlorophyllcontent with increased incubation time in BA-treated leaf segmentsis caused by inactivation of BA either in the leaves or in solution.Of 25 L-amino acids at 50 mM added singly with BA, 19 were antagonisticto the effect of BA on chlorophyll content, but four, L-arginine,L-histidine, L-lysine and L-methionine, significantly enhancedthe effect of BA. The effects of amino acids decreased witha decrease in the amino acid concentration or an increase inBA concentration. When concentrations were decreased to 5 mM,only L-lysine slightly increased the chlorophyll content inBA-treated leaf segments. L-Proline and L-tryptophan did notmodify the effect of BA on chlorophyll content. Effects of aminoacids on chlorophyll content in leaf segments without BA treatmentare similar to those with BA. Furthermore, cycloheximide (50µg/ml), which decreased the soluble amino acid content,increased the chlorophyll content in BA-treated leaf segments. Since an accumulation of soluble amino acids precedes the degradationof chlorphyll in BA-treated leaf segments, probably antagonismscaused by the amino acids accumulated in the tissue, preventthe added BA from being fully effective in stopping chlorophyllloss. (Received June 2, 1980; )     

Responses to sucrose and glutamine by soybean embryos grown in vitro

Immature soybean (Glycine max [L.] Merr. cv. Ransom) embryos were grown in vitro in the presence of different concentrations of sucrose and glutamine to examine how availability of carbohydrate and nitrogen affects dry matter accumulation and embryo composition. Embryos were transferred to fresh medium every 4 days to maintain sucrose and glutamine concentrations of the culture medium. In all experiments, accumulation of dry matter and protein content increased when the sucrose concentration of the culture medium was increased from 1.5 to 150 mM: however, a relatively greater enhancement of dry matter than of protein accumulation resulted in a lower protein concentration at 150 than at 1.5 mM sucrose. Both content and concentration of protein were increased by the increases in glutamine supply to concentrations exceeding 68% protein at 120 mM glutamine. In combination with 150 mM sucrose, however, oil increased as glutamine supply was increased from 0.6 to 6 mM and then decreased as glutamine supply was increased from 6 lo 120 mM. Varying the concentration of sucrose available during seed development also affected embryo composition. Decreased availability of sucrose during either the early or late portion of the culture period resulted in lower accumulation of dry mailer as well as oil. Protein concentration was actually higher for embryos transferred from 150 to 1.5 nM sucrose than for those remaining in 150 mM throughout the culture period: however, the greater percentage of protein was due lo a decrease in accumulation of dry weight. In addition, embryo composition was affected by altering the availability of glutamine during culture, indicating that variation in the level of nitrogen assimilate delivered during seed development can change embryo composition. Decreasing the glutamine concentration of the medium lowered both protein and oil content. In contrast, increasing the glutamine concentration of the medium from 0.6 to 6 mM 8 days after initiation of culture increased the protein content and concentration of the embryo while oil content was not affected.