The differential effect of N6-benzyl-adenine and N6-(Δ2-isopentenyl)-adenine on in vitro propagation of Paeonia suffruticosa Andr. is correlated with different hormone contents

Summary N⁶-benzyl-adenine (BA) enhanced phyllogenesis and axillary bud development of Paeonia suffruticosa during in vitro culture allowing good propagation while N⁶-(²isopentenyl)adenine (iP) did not. During the first five days of culture, the mitotic activity of BA-treated explants was higher than in the iP-treated ones. High BA levels were detected in the BA-treated explants, and this was correlated with the absence of or the low indole-3-acetic acid (IAA) content. The low iP levels measured in iP-treated explants were correlated with high endogenous IAA content; the new cytokinin / auxin ratio could explain the lack of axillary buds and the development of only one leaf. Abscisic acid (ABA) was detected neither in the controls nor in the cytokinin-treated explants during the first week. However, intensive restoration of ABA accumulation was observed in controls from the third week onwards. Both BA and iP-treated explants accumulated less ABA than the controls but this hormone appeared later in the BA-treated explants than in the iP-treated ones.Abbreviations ABA abscisic acid - BA N⁶-benzyl-adenine - BHT butyl-hydroxy-toluene - ELISA enzyme linked immunosorbent assay - FM fresh mass - HPLC high performance liquid chromatography - IAA indole-3-acetic acid - iP N⁶-(²-isopentenyl)adenine - MI mitotic index - 9RBA 9-ß-D-ribofuranosyl-BA - 9RiP 9-ß-Dribofuranosyl-iP - 9RZ 9-ß-D-ribofuranosyl-zeatin - Z zeatin     

Micromorphological studies of adventitious bud formation on Picea abies embryos treated with cytokinin

Embryos of Picea abies (L.) Karst were pulse-treated with water or cytokinin for 2 h and then cultured on medium lacking cytokinin. Adventitious buds developed on cytokinin-treated embryos, but not on water-treated embryos. The general appearance and the surface morphology were similar on water and BA (benzyladenine)-treated embryos after 3 days. The epidermal cells were elongating after 6 days on water-treated embryos, while they were dividing on cytokinin-treated embryos. Furthermore, the cells surrounding the stomata had started to proliferate on BA-treated embryos. This was the first micromorphological sign of bud initiation. During the second week prominent meristemoids developed from these cells. A stoma was observed on the top of each meristemoid. The variation in developmental pattern of meristemoids among different embryos as well as within each embryo was small. However, during the subsequent development of bud primordia and buds, the morphological variation was significant. The meristemoids continued to develop into cone-shaped bud primordia, which successively changed shape during the transition to adventitious buds. The epidermal cells divided and the epidermis did not rupture during the formation of adventitious bud primordia. The epidermis was identified as the protoderm of the bud primordium.     

Changes of Endogenous Hormone Contents During Floral Bud and Vegetative Bud Differentiation in Thin Cell Layer Culture of Cichorium intybus L. Explant

The sectioned thin cell layers (TCL) of flower stalk of Cichorium intybus L. were cultured in MS medium supplemented with NAA and BA or IAA and BA where floral and vegetative buds were developed from the explant. Endogenous IAA, DHZ+DHZR, iPA increased significantly during the floral bud formation, while Z+ZR remained changed. The levels of cytokinins, DHZ +DHZR, iPA, and Z-f-ZR all increased significantly during the vegetative bud formation, however IAA level was reduced during the first 7 days of culture and increased to two-thirds of initial values on the day when the bud primordia were formed. The results suggested that the initiation of floral buds was associated with a high IAA/CTK ratio, whereas the induction of vegetative bud differentiation was related to a low IAA/CTK ratio.     

Polyamine conjugate levels and ethylene biosynthesis: inverse relationship with vegetative bud formation in tobacco thin layers

The effects of two inhibitors of polyamine (spermidine and spermine) biosynthesis, cyclohexylamine (CHA; 5 and 10 mM) and methylglyoxal(bis-guanylhydrazone) (MGBG; 0.1, 0.5 and 1 mM), on the organogenic response in vegetative bud-forming tobacco (Nicotiana tabacum L. cv. Samsun) thin layer explants were evaluated micro- and macroscopically at different times during culture. The final number of buds formed and the percentage of organogenic explants was significantly reduced by both inhibitors, but much more so by MGBG than CHA. This inhibitory effect was already evident in MGBG-treated explants on day 5, in terms of the number of meristemoids per explant. On the contrary, in the presence of CHA, the number of meristemoids on day 5 was higher than that in the controls. Between days 9 and 13, meristemoid formation slowed down considerably in inhibitor-treated explants compared with controls. On day 13, the number of bud primordia was similar in control and CHA-treated explants, but significantly lower in MGBG-treated explants. This inhibitor also induced peculiar cytohistological events, such as a reduced formation of oval-shaped cell aggregates on the explant surface and more frequent cases of nucleolar extrusion, while CHA led to the appearance of hypertrophic epidermal cells; callus formation at the basal end of the explant and xylogenesis were also affected by the inhibitors. Ethylene biosynthesis, measured as [ C]methionine incorporation, was stimulated 2- (day 2) to 3-fold (15 h) by 0.5 mM MGBG, whereas CHA (10 mM) had little effect and aminoethoxyvinylglycine (AVG; 0.1 μM), an ethylene synthesis inhibitor, was strongly inhibitory. In control explants, the incorporation of labelled methionine into ethylene and spermidine followed an inverse trend up to day 8. In these explants, free putrescine increased 32-fold and spermidine increased about 10-fold between days 0 and 8. Trichloroacetic acid (TCA)-soluble conjugated putrescine also accumulated dramatically during culture. While CHA provoked a decline in spermidine levels, MGBG caused an unexpected increase in free spermidine and spermine titres; however, its most conspicuous effect was on the further enhancement of putrescine conjugate accumulation, while CHA and AVG had the opposite effect. Results are discussed in view of establishing a putative link between MGBG-enhanced ethylene synthesis, increased conjugate titres and inhibition of meristemoid formation.     

Nitric oxide mediates the indole acetic acid induction activation of a mitogen-activated protein kinase cascade involved in adventitious root development

Recently, it was demonstrated that nitric oxide (NO) and cGMP are involved in the auxin response during the adventitious rooting process in cucumber (Cucumis sativus; Pagnussat et al., 2002, 2003). However, not much is known about the complex molecular network operating during the cell proliferation and morphogenesis triggered by auxins and NO in that process. Anatomical studies showed that formation of adventitious root primordia was clearly detected in indole acetic acid (IAA)- and NO-treated cucumber explants, while neither cell proliferation nor differentiation into root primordia could be observed in control explants 3 d after primary root was removed. In order to go further with signal transduction mechanisms that operate during IAA- and NO-induced adventitious root formation, experiments were designed to test the involvement of a mitogen-activated protein kinase (MAPK) cascade in that process. Cucumber explants were treated with the NO-donor sodium nitroprusside (SNP) or with SNP plus the specific NO-scavenger cPTIO. Protein extracts from those explants were assayed for protein kinase (PK) activity by using myelin basic protein (MBP) as substrate in both in vitro and in-gel assays. The activation of a PK of approximately 48 kD could be detected 1 d after NO treatment with a maximal activation after 3 d of treatment. In control explants, a PK activity was detected only after 4 d of treatment. The MBP-kinase activity was also detected in extracts from IAA-treated explants, while no signal was observed in IAA + cPTIO treatments. The PK activity could be inhibited by the cell-permeable MAPK kinase inhibitor PD098059, suggesting that the NO-dependent MBP-kinase activity is a MAPK. Furthermore, when PD098059 was administered to explants treated with SNP or IAA, it produced a delay in root emergence and a dose-dependent reduction in root number. Altogether, our results suggest that a MAPK signaling cascade is activated during the adventitious rooting process induced by IAA in a NO-mediated but cGMP-independent pathway. The activation of MAPKs is discussed in relation to the cell responses modulating mitotic process.     

In vitro morphogenesis of organogenic nodules derived from <Emphasis Type="Italic">Sclerocarya birrea</Emphasis> subsp. <Emphasis Type="Italic">caffra</Emphasis> leaf explants

Sclerocarya birrea (marula) is an indigenous South African tree with highly valued medicinal and nutritional properties. Induction of nodular meristemoids from leaf explants was achieved on Murashige and Skoog (MS) and woody plant medium (WPM) supplemented with 6-benzyladenine (BA) in combination with naphthalene acetic acid (NAA), indole-3-butryric acid (IBA) and indole-3-acetic acid (IAA). Induction of nodular meristemoids from 86% of the leaf cultures was achieved on MS medium with 4.0 μM BA and 1.0 μM NAA. High levels (78–100%) of induction were also achieved on WPM with different concentrations of BA (1.0–4.0 μM) and IBA (1.0–4.0 μM). The highest conversion of meristemoids into shoots was only 22% for 4.0 μM BA and 1.0 μM NAA on MS initiation medium. This was improved to 62% when nodular clusters were cultured in a MS liquid medium. Histological studies revealed the globular stage of the nodular meristemoids. This protocol has potential for application in mass micropropagation and plant breeding of S. birrea.     

Histo-Cytological Observations on Callus and Bud Formation in Cultures of Nicotiana tabacum L.

Leaf explants of tobacco were cultured on MS medium supplemented with 2 mg/ l NAA and 0.5 mg/l BA for induction of callus formation, or supplemented with 2 mg/l BA for bud formation. Histocytological observations on callus and bud formation were carried out. Three days after cultivation, mesophyll cells enlarged, the nuclei became more apparent and dark stained, and starch accumulated in the cells. Cell divisions began in the mesophyll cells at the cut ends, in the palisade cells near the vascular bundles and in the vascular parenchyma. Mitotic activity then spreaded over tbc explants, and was most active at the edges of leaf explants. Regular rows of cells appeared as a result of series of transverse divisions in the palisade. The number of chloroplast in the mesophyll cells decreased and degenerated gradually. A number of meristemoids ware initiated in the cultured leaf explants after 7 days of cultivation. They were originated from two kinds of tissues, the mesophyll and vascular bundle, including the phloem parenchyma and vascular sheath. On the medium with NAA and BA, callus formation was induced with vigorous divisions, whereas bud primordia were differentiated from the meristomoids on the medimn with 2 mg/l BA. The buds were developed from both the superficial meristemoids and the meristematic regions deep within the callused leaf explants. The accumulated starch in the cells gradually disappeared as bud formation proceeded.     

Oligogalacturonides enhance cytokinin-induced vegetative shoot formation in tobacco explants, inhibit polyamine biosynthetic gene expression, and promote long-term remobilisation of cell calcium

Long-sized oligogalacturonides (OGs) are cell wall fragments that induce defence and developmental responses. The Ca²⁺-dependent “egg-box” conformation is required for their activity, and polyamines may prevent them from adopting this conformation. Although OGs are known to inhibit auxin-induced growth processes, their effect on cytokinin-induced ones requires investigation. In the present work OGs were shown to promote cytokinin (benzyladenine, BA)-induced vegetative shoot formation from tobacco leaf explants, independent of the presence of CaCl₂ in the medium and of auxin (indoleacetic acid, IAA) supply. The effect of polyamines, putrescine (PU) and spermidine (SD) supplied with/without their biosynthetic inhibitors (DFMO, CHA) was also investigated, and showed that spermidine enhanced adventitious vegetative shoot formation, but only on medium containing Ca²⁺ and IAA. Treatments with inhibitors blocked this promotive effect. OGs did not alter free polyamine concentrations, but caused a moderate increase of conjugated ones, and exhibited an early inhibitory effect on polyamine biosynthetic gene expression. OGs, but not SD, caused long-term changes in calcium-associated epifluorescent signals in the cell walls, and, later, inside the cells of specific tissues. Electron microscopy analysis (ESI system) demonstrated that calcium accumulated in the cell walls and vacuoles of OG-cultured explants. The relationship between OGs, cytokinin, calcium, and polyamines in adventitious vegetative shoot formation is discussed.     

Exogenous Calcium Enhances the Formation of Vegetative Buds,Flowers and Roots in Tobacco Pith Explants Cultured in the Absence of Exogenous Hormones

Pith explants excised from the apical stem internodes of vegetative, flowering, and fruiting tobacco plants were cultured on hormone-free medium in the presence or absence of CaCl₂ (3 mM). The aim was to determine the role of exogenous calcium (Ca²⁺), applied at the concentration normally present in the Murashige and Skoog (1962) medium, in organ formation obtainable in the absence of the exogenous hormonal input. Exclusive formation of vegetative buds was obtained from explants excised from vegetative plants (pure vegetative programme); vegetative buds and flowers (and occasionally roots) on the same sample were obtained from explants from flowering plants (mixed flowering programme); whereas roots, very occasionally associated with vegetative buds and flowers on the same sample, were obtained from explants from fruiting plants (mixed rooting programme). Histological analysis showed that the organs always exhibited indirect regeneration. Exogenous Ca²⁺ promoted the formation of meristemoids and the first phases of their growth into organs, but did not change the realization of the organogenic programme and did not affect callogenesis. Instead, the influence of exogenous Ca²⁺ changed with the programme, when considering the last phases of organ growth (i.e., macroscopic development and elongation), and the appearance of morphological anomalies in the organs.     

Long-sized oligogalacturonides inhibit,whereas spermidine enhances,xylogenesis in tobacco leaf explants

Abstract

Long-sized oligogalacturonides (OGs) are plant cell wall fragments involved in defence responses and developmental processes. A hormone/OG interaction in the control of different organogenic processes is known. However, hormones also modulate polyamine (PA) effects on organogenesis. Furthermore, OGs are known to affect mitotic activity leading to specific morphogenic events, and PAs are known to affect mitotic activity leading to xylogenesis. Thus, it may be reasonable to assume that OGs and PAs affect mitotic activity in the same cell types, and in the same hormone-induced morphogenic processes, e.g., xylogenesis. To gain further insight into this aspect, the effects of OGs, and of putrescine (Put) and spermidine (Spd), on auxin (indoleacetic acid, IAA) plus benzyladenine (BA)-induced morphogenesis in tobacco leaf explants were investigated histologically. The effect of PA biosynthetic inhibitors in the culture medium was also monitored, as well as the combined application of the inhibitor with the corresponding PA. Results show that vascular mitoses consistently occurred in the control (IAA+BA-treated) explants, leading exclusively to xylogenic nodule formation. The application of OGs resulted in an inhibition of vascular mitoses, and into a strong reduction of vascular nodule formation. By contrast, Spd enhanced both vascular mitoses and nodule formation, and Put was less effective than Spd on both events. Taken together, the results reveal a new biological activity of OGs and Spd in morphogenesis, obtained under the same hormonal conditions, and in the same tissue (i.e., the vascular parenchyma), namely the inhibition of xylogenesis by OGs, and its promotion by Spd. The fact that the effects of Spd and OGs on this morphogenic event may involve a different relationship with auxin is discussed.     

Histochemical study of vegetative and floral bud formation in tobacco stem expiants

Primary explants from the inflorescence stem of tobacco and primary explants from the stem of vegetative plants, cultivatedin vitro under the same conditions, display different morphogenetic ability. The former give rise mostly to floral buds, whereas the latter exclusively to vegetative ones. Histological and histochemical analyses of both original andin vitro cultivated explants were made. They showed differences in chlorophyll content and alcohol dehydrogenase (AD) activity of the original explants reflecting their different metabolic status. Bud primordia were initiated in the superficial meristematic layer derived from epidermal tissues. Floral or prefloral apices were characterized by a strong AD activity in all cells of the meristem, while in vegetative apices AD activity was restricted to their uppermost parts. A high rate of procambium differentiation connected with leaf primordia formation was typical of vegetative buds. A higher concentration of glucose (5 %) enhanced cell division in explants, which is also correlated with a higher AD activity. The significance of vascular tissues for differentiation of vegetative buds is discussed. Presented at the International Symposium “Plant Growth Regulators” held on June 18-22, 1984 at Liblice, Czechoslovakia.     

The protein of rolB gene enhances shoot formation in tobacco leaf explants and thin cell layers from plants in different physiological states

The objective was to determine whether the protein of rolB affects shoot formation and whether this potential relationship depends on the developmental stages of the plant and/or on the culture conditions. Thin cell layers (TCL) and leaf explants were excised from tobacco plants in the vegetative and flowering stages and cultured under various hormonal conditions. In TCLs of vegetative-stage plants, the expression of rolB enhanced the formation of the shoot buds under hormone-free conditions and with specific concentrations of auxin and/or cytokinin. Histological examination showed that the induction of the shoot meristemoids was particularly enhanced by rolB protein and that meristemoid growth was accelerated. In leaf explants from vegetative-stage plants, the expression of rolB increased the formation of shoot buds in the presence of 1 M IAA plus 1 or 10 M cytokinin. With BA alone, at a 0.1 M concentration, shoot formation occurred in the transgenic explants only, whereas with concentrations ranging from 0.5 to 10 M, it was higher in these explants than in controls.RolB protein enhanced the formation of shoot buds in TCLs from flowering plants under all hormonal conditions. In the presence of 1 M IAA and kinetin, the protein also increased the flowering response. In leaf explants from flowering plants, the expression of rolB increased the number of shoot buds in the presence of 1 M IAA with 10 M BA.In conclusion, rolB protein promotes shoot formation; it seems to have a positive interaction with cytokinin and an effect on the induction of the meristematic condition.     

De novo root formation in thin cell layers of tobacco: changes in free and bound polyamines

Thin cell layers excised from tobacco ( Nicotiana tabacum L. cv. Samsun) stem internodes, with an appropriate exogenous hormonal balance, were able to form a greater number of roots, and in a larger percentage of the explants (93%) than when they were excised from pedicels (40%). The developmental sequence of root formation and explant growth were followed by histological analysis. Free and bound [trichloroacetic acid (TCA)-soluble and -insoluble] putrescine and spermidine increased in the explants, particularly when root meristemoids appeared. These meristemoids originated in the superficial (day 6 in culture) or deep (days 10–11) layers and inside the newly formed callus (day 25). At those times, TCA-soluble and, to a lesser extent, TCA-insoluble bound putrescine predominated over the other polyamines. Spermine was always present in trace amounts. Polyamines decreased again when root and callus formation was completed (day 30). The involvement of these three classes of polyamines (free, TCA-soluble and -insoluble) in morphogenic processes is discussed.     

菊苣薄层培养花芽,营养芽分化中内源激素的动态变化

菊苣（Ｃｉｃｈｏｒｉｕｍ ｉｎｔｙｂｕｓＬ．）花梗薄层细胞培养于ＭＳ附加ＮＡＡ 和ＢＡ 或ＩＡＡ 和ＢＡ 的ＭＳ培养基上有花芽或营养芽分化． 花芽分化中内源ＩＡＡ、ＤＨＺ＋ ＤＨＺＲ、ｉＰＡ 含量明显增加,而Ｚ＋ ＺＲ变化不明显．营养芽分化中内源细胞分裂素含量增加明显,而ＩＡＡ 在培养前７ ｄ 含量下降,随后有所增加,在原基形成时含量达原初水平的２／３． 可见,花芽分化比营养芽分化所需内源ＩＡＡ／ＣＴＫ 比值要高     

Calcium and calcium-dependent protein kinases are involved in nitric oxide- and auxin-induced adventitious root formation in cucumber

A few years ago it was demonstrated that nitric oxide (NO) and cGMP are involved in the auxin response during adventitious root (AR) formation in cucumber (Cucumis sativus). More recently, a mitogen-activated protein kinase cascade was shown to be induced by IAA in a NO-dependent, but cGMP-independent, pathway. In the present study, the involvement of Ca2+ and the regulation of Ca2+-dependent protein kinase (CDPK) activity during IAA- and NO-induced AR formation was evaluated in cucumber explants. The effectiveness of several broad-spectrum Ca2+ channel inhibitors and Ca2+ chelators in affecting AR formation induced by IAA or NO was also examined. Results indicate that the explants response to IAA and NO depends on the availability of both intracellular and extracellular Ca2+ pools. Protein extracts from cucumber hypocotyls were assayed for CDPK activity by using histone IIIS or syntide 2 as substrates for in-gel or in vitro assays, respectively. The activity of a 50 kDa CDPK was detected after 1 d of either NO or IAA treatments and it extended up to the third day of treatment. This CDPK activity was affected in both extracts from NO- and IAA-treated explants in the presence of the specific NO-scavenger cPTIO, suggesting that NO is required for its maximal and sustained activity. The in-gel and the in vitro CDPK activity, as well as the NO- or IAA-induced AR formation, were inhibited by calmodulin antagonists. Furthermore, the induction of CDPK activity by NO and IAA was shown to be reliant on the activity of the enzyme guanylate cyclase.     

<Emphasis Type="Italic">In vitro</Emphasis> culture of <Emphasis Type="Italic">Hypericum rumeliacum</Emphasis> Boiss. and production of phenolics and flavonoids

The Balkan endemic species, Hypericum rumeliacum, Guttiferae was introduced in vitro for the first time with the aim to study the type of morphogenetic response to plant growth regulators and ability to produce phenolics and flavonoid compounds. The morphoregulatory effect of 2,4-dichlorophenoxyacetic acid (2,4-D), 1-naphtaleneacetic acid (NAA), 6-benzyladenine (BA) and combination of BA with NAA in Murashige–Skoog's basal medium on leaf lamina, internode stem segment, stem node and root cuttings was studied. Histological analysis of the structures regenerated from the primary explants proved the presence of both, embryoids and meristemoids. The node explants cultivated on BA-supplemented medium were the most favourable for regeneration through meristemoids. Therefore a double-stage culture approach, allowing an effective multiplication of large quantities of plant shoots in vitro along with maintenance of the biosynthetic capacity of the culture was developed. It comprised one subculture of three-nodal stem explants derived from the stock shoot cultures on MS medium supplemented with 0.2 mg/l BA followed by subculture of the induced multiple shoots on cytokinin-free MS medium. Determination of the total phenolics and flavonoids showed that the decrease of the levels of these secondary metabolites is transitional, as the exclusionof BA from the medium resulted in an increase of their total content.     

金桔组织培养中愈伤组织和芽形成的组织细胞学观察

用金桔茎段为外植体,培养在附加1.0毫克/升BA和0.l毫克/升IBA的MS培养基上,诱导愈伤组织和芽形成。观察了愈伤组织和芽形成过程中的组织细胞学变化。培养一周后,在茎组织切口两端开始膨大,细胞增大和开始分裂。培养两周后,开始形成瘤状愈伤组织。在愈伤组织中有形成层状分生组织、维管组织结节和分生细胞团。培养四周后,表层的分生细胞团分化形成大量芽原基,同时愈伤组织深层也出现分生细胞团。带节茎段可从切口两端的愈伤组织分化形成芽,亦可从叶腋的潜伏芽直接形成芽。     

Inhibition of adventitious root development in apple rootstocks by cytokinin is based on its suppression of adventitious root primordia formation

Cytokinin (CK) inhibits adventitious root (AR) formation in stem cuttings. Little is known, however, about the mechanism underlying the inhibitory effect. In this study, 2 mg l^?1 of exogenous 6‐benzyl adenine (6‐BA) was administered to 3 and 7‐day‐old apple rootstocks ‘M.26’ cuttings (3 and 7 days 6‐BA) by transferring them from a rooting medium containing indole‐3‐butanoic acid to the medium containing 6‐BA. Anatomical and morphological observations revealed that the exogenous application of 6‐BA inhibited primordia formation in the 3 days 6‐BA but not the 7 days 6‐BA group. The concentration of auxin (IAA), the ratios of IAA/CK and IAA/abscisic acid were lower in 3 days 6‐BA than in 7 days 6‐BA. Expression analysis of genes known to be associated with AR formation was also analyzed. In the 3 days 6‐BA group, high level of CK inhibited the synthesis and transport of auxin, as a result, low endogenous auxin level suppressed the auxin signaling pathway genes, as were other AR development and cell cycle related genes; all of which had an inhibitory impact on AR primordium formation. On the contrary, low CK level in the 7 days 6‐BA, reduced the inhibitory impact on auxin levels, leading to an upregulated expression of genes known to promote AR primordia formation. Collectively, our data indicated that 3–7 days is the time period in which AR primordia formation occurs in cuttings of ‘M.26’ and that the inhibition of AR development by CK is due to the suppression of AR primordia development over 3–7 days period after culturing in rooting medium.     

High Frequency of Shoot Regeneration from Hypocotyls and Stem Segments of Antirrhinum majus (Snapdragon)

A broadly applicable direct shoot regeneration method from hypocotyls and stem explants has been developed for six cultivars of Antirrhinum majus L. In order to establish a stable and high frequency of shoot regeneration system, leaves, hypocotyls and stem explants of six cultivars were tested with 72 combinations of auxin (naphthaleneacetic acid (NAA) or 3-indoleacetic acid (IAA)) and cytokinin (6-benzylaminopurine (BA) or zeatin (Z)). A few adventitious shoots were directly regenerated from hypocotyl segments of cv. Orchid on MS medium with NAA + BA, IAA + BA, NAA + Z and IAA + Z. High frequency of direct shoot regeneration was obtained from hypocotyl segments on MS medium with 0.05, 0.1 or 0.25 mg l⁻¹ NAA + 2 mg l⁻¹ Z and 0.5 mg l⁻¹ IAA + 2 mg l⁻¹ Z. Finally, stable and high frequency (92–100%) of shoot regeneration with more than 10 adventitious shoots per explant was achieved from the hypocotyls and stem explants of all six cultivars on MS medium with 0.25 mg l⁻¹ NAA + 2 mg l⁻¹ Z. The shoots emerged directly from the hypocotyls and stem segments 4 weeks after culture initiation.     

Calcium Transport in Membrane Vesicles Isolated from Maize Coleoptiles (Effect of Indoleacetic Acid and Fusicoccin)

Maize (Zea mays L.) coleoptile segments loaded with 45Ca released about 50% of the ion after 1 h when treated with indoleacetic acid (IAA). In contrast, fusicoccin (FC) had no effect. The same relation was found when ATP-dependent Ca2+ transport, measured as 45Ca uptake, was determined in a plasmalemma-rich membrane vesicle fraction isolated from coleoptiles treated or untreated for 1 h with IAA or FC. In fact, IAA-treated membranes showed an increase in ATP-dependent 45Ca uptake by more than 30% with respect to the control and the FC treatment. Ca2+ uptake in IAA-treated membranes was only slightly affected (+27%) by supplying calmodulin (Cam) exogenously. However, Ca2+ uptake in membranes from the control and FC-treated coleoptiles were stimulated (+80%) by exogenous Cam. Calmidazolium, a Cam antagonist, inhibited Ca2+ uptake in the IAA treatment (-48%) to a greater extent with respect to the control and FC treatment (-33 and -29%, respectively). A possible relationship between the effect of IAA on the ATP-dependent Ca2+ transport activity, the involvement of Cam, and their effect on growth are discussed.