Adventitious shoot formation and plant regeneration from tissues of tomatillo (Physalis ixocarpa Brot.)

Cultured hypocotyl explants of tomatillo (Physalis ixocarpa Brot.), were evaluated with regard to their morphogenic responses to combinations of benzyladenine (BA, 0–5 M) with either naphthaleneacetic acid (NAA, 0–50 M) or 2,4-dichlorophenoxyacetic acid (2,4-D, 0–50 M). The induction of shoots or roots was dependent on the cytokinin/auxin combination.Hypocotyl explants failed to form shoots when they were grown on media containing either a cytokinin or an auxin alone. The highest frequency of shoot formation was observed on media containing 12.5–25 M BA and 5 M NAA. Likewise the highest frequency of root formation was observed on media supplemented with 1 M BA and 1 M NAA. Complete plants were regenerated and transferred to soil, where they reached maturity.     

Somatic embryogenesis and plant regeneration from zygotic embryo culture in blue spruce (Picea pungens Engelman.)

Summary Somatic embryogenesis and plantlet formation have been achieved from cultured mature zygotic embryos of blue spruce (Picea pungens Engelman.). The effect of three basal media LP, LM, and BLG, all used at half-strength, was tested at the induction phase. LM medium induced somatic embryogenesis to a higher extent than LP whereas BLG did not produce any embryonal-suspensor mass representing stage 1 somatic embryos. The embryonal-suspensor mass was induced on a wide range of auxin/cytokinin ratios. However, media containing either 2 M NAA and 10 M BA, or 10 M NAA and 5 M BA produced somatic embryos that gave the highest frequency of plantlets. The level of ABA required in the maturation medium for somatic embryos to mature properly varied with the auxin/cytokinin levels in the induction medium on which the somatic embryos were derived. Inclusion of AgNO₃ (10 – 100 M) in the induction medium reduced somatic embryogenesis and embryo conversion.Abbreviations NAA naphthalene-acetic acid - BA N⁶-benzylaminopurine - ABA abscisic acid     

Rapid clonal propagation of guava through in vitro shoot proliferation on nodal explants of mature trees

In vitro clonal propagation of guava Banaras local was achieved by culturing nodal explants of mature trees on Murashige and Skoog (MS) revised medium supplemented with 4.5 M 6-benzyladanine (BA) alone or in combination with either 0.6 M indole-3-acetic acid (IAA), 0.5 M indole-3-butyric acid (IBA) or 0.3 M gibberellic acid (GA₃). Multiple shoots were induced to form by enhancement of axillary branching and BA (4.5 M) without any auxin and gibberellin was found to give best shoot multiplication rate. In this medium 3–6 shoots developed on explants collected from field-grown plants and 5–10 shoots developed on explants taken from in vitro proliferated shoots within 12 wk of culture. A prior transfer of shoot clumps to a medium containing a lower concentration of BA (0.5 M) before harvesting of cuttings for rooting allowed rapid extension growth and increased the number of usable shoots per culture. Adventitious rooting occurred after subculturing excised shoots on a medium containing 1/2 strength MS salts, 1.5% sucrose, 1 M each of IBA and -naphtha-leneacetic acid (NAA), and 1 gl^-1 activated charcoal. Regenerated plantlets were successfully established on soil.     

Plant development in long-term embryogenic callus lines of Cucurbita pepo

Embryogenic callus derived from pumpkin hypocotyl segments was induced and maintained for 15 years on MS medium supplemented with the auxins IBA (4.9 M), 2, 4-D (4.5 M) or IAA (5.7 M). On induction media continued embryo maturation and development of adult plants typically failed. Therefore, small embryogenic clumps and individually isolated embryos were subcultured two to four times on one of the conversion media: MS supplemented with 1.5% sucrose and (a) no hormone, (b) 2.9 M IAA, (c) 5.7 M IAA, (d) 11.4 M IAA, (e) 12 M IEt, (f) 3.8 M ABA or (g) 2% activated charcoal. The cell line and the kind of auxin used in the induction and maintenance medium, both had a marked influence on the development of plantlets. The best result was achieved with a line that has been induced and maintained for 15 years on MS with IBA. In the IBA line, out of 100 embryos, 77 developed into plantlets on MS medium supplemented with 11.4 M IAA.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - BA 6-benzylaminopurine - ABA abscisic acid - 2, 4-D 2, 4-di-chlorophenoxyacetic acid - IEt indole-3-etha-nol - MS Murashige and Skoog (1962) medium     

Somatic embryogenesis in cultured mature zygotic embryos of Abies balsamea

Embryo suspensor masses (ESMs) were induced by culture of isolated mature zygotic embryos of balsam fir [Abies balsamea (Mill.)] on media containing 10 M cytokinin [6-(--dimethylallylamino)purine (2iP), 6-benzyladenine (BA), or thidiazuron (TDZ)]. Once induced, ESMs proliferated on media containing 2iP, BA or TDZ (10 M) or on 4.5 M BA in combination with 10 M naphthyl-1-acetic acid. When ESMs were transferred to media containing 5–80 M abscisic acid, cotyledonary-stage embryos were formed. Embryos were readily germinated on medium lacking growth regulators.Abbreviations ABA abscisic acid - BA 6-benzyladenine - ESM embryo-suspensor mass - 2iP 6-(--dimethylallylamino)purine - NAA naphthyl-1-acetic acid - TDZ N-phenyl-N-1,2,3-thiadiazol-5-yl urea (thidiazuron)     

Do Casuarina cunninghamiana seedlings dependent on symbiotic N2 fixation have higher phosphorus requirements than those supplied with adequate fertilizer nitrogen?

P requirements of Casuarina cunninghamiana seedlings inoculated with Frankia and reliant on symbiotic N2 fixation were compared with those of uninoculated seedlings grown with 4 levels of fertilizer N in a solution culture system.Growth responses to increasing P supply depended on the N treatment that had been imposed. At the two lowest levels of N supply (0 t M and 100 t M) growth was relatively poor and there was no response to increasing P supply above 10 t M and 50 t M P respectively. In contrast, inoculated seedlings and those with higher levels of combined N (500 t M and 1000 t M) had significantly greater shoot dry weights (except at 0.1 t M P), and they responded to increasing P supply to between 50 and 100 t M P. At each level of P supply, the dry weights of seedlings in these 3 N treatments were similar. Nitrogen concentrations in shoots of seedlings provided with fertilizer N decreased with increasing P supply to 50 t M and then remained unchanged. In contrast, N concentrations in shoots of inoculated seedlings increased with increasing P supply to 25 t M P. At the 2 highest levels of P supply, N concentrations in shoots of inoculated plants were significantly higher than those in seedlings provided with fertilizer N. In all N treatments, P concentrations in shoots increased with increasing P supply; concentrations were similar in inoculated seedlings and those with the 2 highest levels of fertilizer N across all levels of P supply.Alleviation of P deficiency in inoculated seedlings increased nodule number, nodule dry weight, N2 fixation g-1 nodule dry weight (nodule 'efficiency'), P concentration in nodules, proportion of total seedling biomass allocated to nodules and average nodule size. However, all these parameters reached their maximum values at levels of P supply at, or below, those required for maximum host-plant growth (50 to 100 t M P).The results indicate that the P requirement for host plant growth per se is similar to, or higher than, that required for symbiotic N2 fixation processes.     

Lateral bud culture of papaya (Carica papaya L.) for clonal propagation

Lateral buds may be preferred to shoot tips for in vitro propagation of papaya because of its unbranched nature. Proliferating shoot cultures from lateral buds appeared extremely compact with shortened internodes and leaf lamina of the cytokinin level (BAP 2 M) reported for multiple shoot production from shoot tips. ZEA (4 M) and 2iP (8 M) although reduced the proliferation rate, resulted in better growth of the shoot from lateral bud. Rooting was observed with IBA 20 M but plantlets so produced remained stunted.     

Plant regeneration via somatic embryogenesis in Styrian pumpkin: cytological and biochemical investigations

Somatic embryo formation was induced from cotyledon explants of Styrian pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) by using a solid MS medium supplemented with 16.11M NAA and 4.44M BA or 26.85M NAA and 13.32M BA. The callus proliferation was more efficient on medium supplemented with 26.85M NAA and 13.32M BA. In contrast, the embryogenic response was higher on medium with lower concentrations of growth regulators (16.11M NAA and 4.44M BA). The time needed for embryo induction did not depend on medium composition. Embryos in globular stage were transferred to three different maturation media, containing 2.89M GA₃ in combination with 0.54M NAA, 11.42M IAA and growth regulator-free medium. The germination rate was the highest when embryos were cultured on medium with 11.42M IAA. Plantlets grown on this medium achieved maturity suitable for transplantation into soil within 9 to 10weeks. The regenerated plants were successfully transferred into field and developed fertile flowers and set fruits. Biochemical analysis showed significant lower total glutathione levels among in vitro grown plantlets compared to seedlings grown in soil. When the plantlets were transferred into soil, they reached a normal size within a month and the glutathione concentration was comparable to seed-derived plants at the same developmental stage. Transmission electron microscopy was used to investigate possible differences in the ultrastructure of cells from callus cultures, and leaf cells of regenerated and seed-derived plants. Differences in the ultrastructure were found within chloroplasts which contained only single thylakoids, large starch grains and small plastoglobuli in callus cells in comparison to leaf cells, which possessed a well developed thylakoid system, small starch grains and large plastoglobuli.     

In vitro regeneration through organogenesis of Meconopsis simplicifolia — An endangered ornamental species

Meconopsis simplicifolia (D.Don) Walp. could be propagated by induction of adventitious shoots from callus produced on hypocotyl, cotyledon and rosette leaf explants of 4-month-old seedlings. Callus was initiated on agar-solidified Murashige and Skoog medium supplemented with 1 M kinetin +10 M -naphthaleneacetic acid (NAA). Shoots formed when the callus was subcultured on medium supplemented with kinetin or benzyladenine (BA) in combination with NAA, indoleacetic acid, indolebutyric acid or gibberellic acid. Excised shoots were rooted on medium containing auxin with 10 M NAA producing the best rooting (55%).Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxy-acetic acid - FAA formalin-acetic acid-alcohol - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA -indolebutyric acid - NAA -naphthaleneacetic acid     

Clonal propagation of Callistemon by tissue culture techniques

Callus development in Callistemon viminalis was readily achieved when axillary buds derived from nodal tissue were placed in a medium containing macro- and micro-nutrients, sucrose (0.06 M), inositol (300 M), nicotinic acid (20 M), pyridoxine hydrochloride (3 M), thiamine hydrochloride (2 M), riboflavin (10 M), cytokinins (5 M) and auxins (0.1 M). The presence of benzylaminopurine (5 M) and p-chlorophenoxyacetic acid (0.1 M) promoted the most vigorous callus development and sprout formation. Rooting of nodal material was rare but occurred readily following the transference of sprouts developed on callus to a basal medium containing sucrose and salts. Root initiation was stimulated, however, by the presence of auxins. Chlorophenoxyacetic acid while stimulating root initiation repressed root growth. Indole butyric acid stimulated both root initiation and shoot growth at concentrations of 0.005 to 0.1 M. The treatment of choice for rooting and shoot growth was the addition of indole butyric acid at a concentration of 0.01 M.     

Plant regeneration from cotyledons of niger

Callus initiation from seedling explants of niger (Guizotia abyssinica Cass) cv. Ootacamund was found to be better on LS medium containing kinetin (1.4 M) plus 2,4-dichlorophenoxyacetic acid (9 M) than its analogues. Embryoids were induced directly from cotyledons on LS medium supplemented with 2,4,5-trichlorophenoxyacetic acid and 2,4,5-trichlorophenoxypropionic acid. When cotyledon-derived callus was subcultured onto medium with 10.7 M naphthalene-acetic acid and 2.3 M kinetin, embryogenesis was observed. Multiple shoots were obtained from cotyledonary explants in presence of MS medium containing 4.4 M benzyladenine and 11.4 M indoleacetic acid. Regenerated plants that were transferred to pots and grown to maturity were morphologically normal and fertile.Abbreviations NAA naphthaleneacetic acid - IAA indoleacetic acid - BA benzyladenine - GA₃ gibberellic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxypropionic acid - 2,4,5-TP 2,4,5-trichlorophenoxypropionic acid - ABA abscisic acid     

Morphological and hormonal characterisation of strawberry vitroplants raised through axillary or stipular adventitious shooting

Adventitious stipular bud formation occurred in vitro in many strawberry cultivars during the proliferation phase onmedium containing Knop macronutrients, MS micronutrients, vitamins, aminoacids,2.22 M BAP, 2.46 M IBA and 0.29M GA₃. As described previously for cultivarGorella,cultivar Elsanta also showed adventitious stipular buds developing on theabaxial median zone between the stipule tips. To compare the shoots producedfrom both types of buds, clonal propagation was initiated from stipular budsandfrom axillary buds on the above mentioned medium. Stipular buds were separatedfrom the meristem-tip initiated plantlet and cultivated in the presence of alower BAP concentration (1.33 M) to prevent further stipularbud formation.During proliferation cycles, stipular originated propagules werevery easily distinguished by their specific leaf phenotype and light greencolour in comparison to plantlets cloned for an axillary bud. Theirmultiplication rate and cytokinin content were also higher than for axillarybuds. No significant difference was observed in auxin content.     

Somatic embryogenesis and plant regeneration from the immature cotyledonary tissues of cultivated tea (Camellia sinensis (L).O. Kuntze)

Embryogenic callus development, plant regeneration, and plant recovery were achieved from immature cotyledon explants of cultivated tea, when cultured on MS basal medium. The somatic embryo induction frequency was influenced when the medium was supplemented with 1 M auxin (NAA, NOA, 2,4-D, TPB, and PBOA) in combination with cytokinin (0.5 M BA, KIN) or 10% CM. The highest somatic embryo induction frequency was obtained using PBOA + BA or PBOA + KIN treatments. All auxins except 2,4-D stimulated rhizogenesis using 0.8% and l.5% agar concentrations, and differentiation of a characteristic swelling and friable callus from the exposed surface of the explant that remained nonembryogenic. Conversely, the novel auxins TPB and PBOA at 1 M concentration with 3% or 6% agar, produced somatic embryo induction, while at 0.8% and 1.5% produced nonembryogenic callus. Explants isolated proximal to the zygotic embryonal axis showed a greater somatic embryo induction frequency than did the distal explants. The embryogenic competence was maintained through repetitive embryogenesis for a period of over 18 months. The somatic embryos developed into plantlets when incubated on hormone-free medium. The conversion frequency was increased by 50% in MS medium containing 1 M Brassin and 0.8% agar. Concentration of agar at 3% and 6% decreased the conversion frequency and promoted anomalous plantlet development. The normal plantlets were treated with 1 M IAN, 1 M Brassin and 10 Phloroglucinol in liquid MS medium for 15 d, where profuse lateral roots were induced favoring a high rate of plant recovery.     

Phenylacetic acid-induced somatic embryogenesis in cultured hypocotyl explants of geranium (Pelargonium x hortorum Bailey)

Summary The effect of a non-indole compound, phenylacetic acid (PAA), on the induction of somatic embryogenesis in tissue cultures of geranium (Pelargonium x hortorum Bailey cv. Scarlet Orbit Improved) was investigated. Hypocotyl explants derived from young, dark-grown seedlings were cultured on Murashige and Skoog (1962) medium (MS) supplemented with PAA or IAA (0.01–120 M) alone or in combination with BAP (8 M). Somatic embryogenesis was induced by both PAA and IAA at 0.01–20 M with 8 M BAP, however, the optima differed considerably for the two compounds. Maximal activity of IAA for somatic embryogenesis was found at 0.1–2.5 M, whereas PAA gave best results at 10 and 20 M under identical culture conditions. Higher concentrations (30–120 M) of IAA or PAA in the medium induced callusing in the explants, but the callus was neither embryogenic nor morphogenic.Abbreviations BAP N⁶-benzylaminopurine - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) - PAA phenylacetic acid     

Multiple Hormonal Control of the Lectin Content in Roots of Wheat Seedlings

The effects of 0.2 M GA, 4.4 M benzyladenine (BA), and 5.7 M IAA on the contents of wheat germ agglutinin (WGA) and ABA in the roots of four-day-old wheat (Triticum aestivumL.) seedlings were studied. All phytohormones tested almost doubled the level of WGA. BA and IAA evidently stimulated the WGA accumulation by inducing ABA accumulation, whereas GA affected the level of the WGA in an ABA-independent way. The authors conclude that phytohormones control the WGA level via several pathways.     

Somatic embryogenesis in Simarouba glauca

Frequency of somatic embryogenesis from callus cultures derived from immature cotyledon explants of Simarouba glauca Linn. was highest on solid MS medium supplemented with 11.1 M benzyladenine and 13.42 M -naphthaleneacetic acid. On transfer of the somatic embryos into maturation medium containing half-strength MS medium supplemented with 1.89 M abscisic acid (ABA) and 2% (w/v)sucrose, 20–25 % of embryos germinated within 20 days of culture with distinct cotyledon, hypocotyl and radicle.     

Hormones andCuscuta development: In vitro induction of haustoria by cytokinin and its inhibition by other hormones

Cytokinins induced haustoria formation in excised 10-mm segments ofCuscuta vine, the subapical 25-to-50-mm region being most responsive, producing a mean of 4–6 haustoria per segment. The order of effectiveness of cytokinins continuously applied (72 h) was 6-benzylaminopurine (BA) isopentenyladenine (iP) zeatin (Z). Ribosides of BA and Z were as effective as the bases, whereas riboside of iP ([9R]iP) was half as effective as iP. Haustoria induction was influenced by weather and seasonal conditions at the time of vine collection; materials obtained on warm, sunny days responded better than those obtained on rainy, cloudy, or cool days. Haustoria were induced equally well all around the segment, and no thigmostimulus was needed for induction. p ]A 10-min pulse of 100 M BA induced half as many haustoria as a 60-min pulse or continuous application of BA. White light inhibited haustoria induction elicited by a short (30-min) pulse of BA, whereas a longer (120-min) BA application overcame this light inhibition. Auxins (IAA or NAA, 1–10 M), gibberellin (GA₃, 1–10 M), ethylene (as ethrel, 10–100 M), and abscisic acid (ABA, 100 M) were individually inhibitory (60–80%) with respect to haustoria induction when given continuously with 50 M BA. A 60-min pulse of auxins (10 M), GA₃ (100 M), or ethrel (10 M), given at various time intervals during or after a 60-min pulse of 100 M BA, showed that inhibition was maximal (70–95%) between 4 and 16 h of BA application and negligible (GA₃) or much reduced (auxin, ethrel) at 20 h, indicating a commitment to haustoria formation by this time.     

Karyometry of nuclei in liver cells

Summary In untreated adult male albino rats nuclear volume and the percentage of binucleate cells were determined in the first layer of hepatocytes adjacent to hepatic venous branches of varying diameters (<40 m, 40 m–80 m, 80 m–120m, 120 m–160 m, >160 m), and in the third and fourth layer of hepatocytes in the remainder of the perivenous parenchyma. In the first layer of hepatocytes adjacent to the vascular structures means of nuclear volume are significantly lower and percentage of binucleate cells significantly higher than in the cells of the remainder of the perivenous parenchyma. Within each area measured distribution curves of nuclear volume classes were homogeneous but showed heterogeneity in comparison with each other. The morphometric data presented in this study strongly support the opinion of the heterogeneity of liver cells in the perivenous zone, as previously postulated on the basis of histochemical investigations.Supported by the Deutsche Forschungsgemeinschaft (Hi 318/2-1)     

Interaction of environment and ABA and GA treatments on the maize anther culture response

Treatments designed to influence abscisic acid (ABA) or gibberellin (GA) concentrations were applied to developing tassels of maize (Zea mays L.) plants in different environments or to anthers in culture to determine the effect on formation of embryo-like structures (ELS). Production of ELS was significantly affected in certain environments when ABA, GA₃, ancymidol, or fluridone solutions were pipetted into whorls of field-grown plants approximately 3 days before tassel harvest. In 1996 anthers from 10 M ancymidol-treated plants were most responsive, producing 35 ELS/100 anthers and 50 M GA₃-treated plants were least responsive, producing 12 ELS/100 anthers. In 1997 under hotter, drier conditions, anthers from 50 M GA₃-treated plants were most responsive, producing 20 ELS/100 anthers and those from 50 M ABA-treated plants were least responsive, producing 2.4 ELS/100 anthers. Anthers from growth chamber plants were significantly more responsive when grown in a 16-h than a 12-h photoperiod. With the 16-h photoperiod the response was significantly greater with a 250 M ABA whorl treatment. With the 12-h photoperiod there was no significant effect from whorl treatments. Modification of the culture medium with added ABA, GA₃, ancymidol, or fluridone was generally ineffective, except in 1997 when the response was significantly higher with 1 M ABA added to the culture medium. The results suggest that the maize anther culture response may be influenced by environmental conditions that interact with ABA and GA treatments to donor plants during tassel development.     

Influence of 2,3,5-triiodobenzoic acid on the transport and metabolism of IAA in lupin hypocotyls

The influence of 2,3,5-triiodobenzoic acid (TIBA) on the transport and metabolism of indolyl-3-acetic acid (IAA) was studied in etiolated lupin (Lupinus albus L) hypocotyls. Double isotope-labeled IAA [(5-³H)-IAA plus (1-¹⁴C)-IAA] was applied to the cut surface of decapitated seedlings. This confirmed that the species mobilized was unaltered IAA and permitted us to measure the in vivo decarboxylation of applied IAA. A pretreatment with TIBA applied to the cut surface produced a partial or drastic inhibition in the basipetal IAA movement at 0.5 or 100 M, respectively. Since TIBA inhibits auxin polar transport by interfering with the efflux carrier, the above results suggest that 100 M TIBA is sufficient to saturate the binding sites in the transporting cells. Compared to the control plants, in vivo decarboxylation of IAA was enhanced in 0.5 M TIBA-treated plants, while no decarboxylation was detected after treatment with 100 M TIBA. The in vitro decarboxylation of (1-¹⁴C)-IAA catalyzed by purified peroxidase was moderately activated by 100 M and unaffected by 0.5 M TIBA. The paradoxical effect of TIBA in vivo vs in vitro assays suggests that the in vivo effect of TIBA on IAA oxidation might be the consequence of the action of TIBA on the auxin transport system. Thus, transport reduction by 0.5 M TIBA caused a temporary accumulation of IAA in that apical region of the hypocotyl which has the highest capacity to decarboxylate IAA. In the presence of 100 M TIBA, a concentration which presumably saturates the efflux carriers, most of the added IAA can be expected to be located in the transporting cells where, according to the present data, IAA decarboxylation cannot take place.