Thin Layer Explants Research Articles

Biologically active oligosaccharides from pectins of Pisum sativum L. seedlings affecting root generation.

Two physiologically active oligosaccharide fractions were isolated from pectin of Pisum sativum L. cell wall after its partial acid hydrolysis. These fractions displayed stimulating and inhibiting effects on root formation in thin-layer explants. The subsequent separation of these fractions by gel permeation and anion-exchange chromatography resulted in fractions with effective concentrations two orders of magnitude lower than the concentrations of the initial fractions. The resulting oligosaccharides displayed their effect on the earliest stage of the rhizogenesis associated with formation of root primordias. The rhizogenesis-inhibiting fraction suppressed cell division by 30-50%. The stimulating fraction mainly contained fragments of xyloglucan and galactan, and the inhibiting fraction contained fragments of xyloglucan, galactan, and arabinan. The polymerization degrees of the stimulating and of the inhibiting oligosaccharides were 10-11 and 5-6, respectively.

Methyl jasmonate upregulates biosynthetic gene expression, oxidation and conjugation of polyamines, and inhibits shoot formation in tobacco thin layers

The effect of methyl jasmonate (MJ) on de novo shoot formation and polyamine metabolism was investigated in thin layer explants of tobacco (Nicotiana tabacum L. cv. Samsun). A relatively low concentration of MJ (0.1 microM) enhanced explant fresh weight, but had no effect on the final number of shoots per explant while higher concentrations (1 and 10 microM) significantly inhibited organogenesis. The histological study revealed that, with increasing concentrations of MJ, the formation of meristemoids and shoot domes declined and the incidence of cell hypertrophy increased. In explants cultured with 0.1, 1 or 10 microM MJ, the endogenous levels of free putrescine, spermidine and spermine generally declined compared with controls, after 7 and 15 d. Perchloric acid-soluble conjugated polyamines accumulated dramatically during culture, but much more so in the presence of MJ than in controls. Acid-insoluble conjugated spermidine alone increased in response to the elicitor. Activities of the putrescine biosynthetic enzymes arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17) in the soluble fraction of MJ-treated explants displayed up to 3-fold increases relative to control explants. However, the most relevant increases in these enzyme activities occurred in the particulate fraction. The activity of S:-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21), an enzyme involved in spermidine and spermine biosynthesis, was also stimulated by exposure to MJ. Northern analyses revealed MJ-induced, generally dose-dependent, increases in the mRNA levels of all three enzymes. Diamine oxidase (DAO, EC 1.4.3.6) activity was stimulated by MJ mainly in the cell wall fraction. The upregulation of polyamine metabolism is discussed in relation to the morphogenic behaviour of MJ-treated explants.

Methyl jasmonate upregulates biosynthetic gene expression, oxidation and conjugation of polyamines, and inhibits shoot formation in tobacco thin layers

The effect of methyl jasmonate (MJ) on de novo shoot formation and polyamine metabolism was investigated in thin layer explants of tobacco (Nicotiana tabacum L. cv. Samsun). A relatively low concentration of MJ (0.1 μM) enhanced explant fresh weight, but had no effect on the final number of shoots per explant while higher concentrations (1 and 10 μM) significantly inhibited organogenesis. The histological study revealed that, with increasing concentrations of MJ, the formation of meristemoids and shoot domes declined and the incidence of cell hypertrophy increased. In explants cultured with 0.1, 1 or 10 μM MJ, the endogenous levels of free putrescine, spermidine and spermine generally declined compared with controls, after 7 and 15 d. Perchloric acid‐soluble conjugated polyamines accumulated dramatically during culture, but much more so in the presence of MJ than in controls. Acid‐insoluble conjugated spermidine alone increased in response to the elicitor. Activities of the putrescine biosynthetic enzymes arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17) in the soluble fraction of MJ‐treated explants displayed up to 3‐fold increases relative to control explants. However, the most relevant increases in these enzyme activities occurred in the particulate fraction. The activity of S‐adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21), an enzyme involved in spermidine and spermine biosynthesis, was also stimulated by exposure to MJ. Northern analyses revealed MJ‐induced, generally dose‐dependent, increases in the mRNA levels of all three enzymes. Diamine oxidase (DAO, EC 1.4.3.6) activity was stimulated by MJ mainly in the cell wall fraction. The upregulation of polyamine metabolism is discussed in relation to the morphogenic behaviour of MJ‐treated explants.

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 [ image 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.

Protease activity, organisation of vegetative primordia and effect of putrescine in tobacco thin layers

Since in tobacco thin layers exogenous putrescine alters the physiological and mor‐phogenic responses induced by IAA (indole‐3‐acetic acid) and/or BA (benzylade‐nine), the effect of this polyamine on protease activity and on the formation of meristemoids and vegetative primordia was studied during morphogenesis. Superficial thin layer explants, excised from the stem of tobacco (Nicotiana tabacum L. cv. Samsun) plants in the vegetative stage, were cultured under various hormonal conditions (IAA, IAA+BA, BA) and in a hormone‐free medium, in the presence or absence of 100 μM putrescine. Histological analysis showed that no meristemoids were formed on the control medium or with putrescine alone and only a few were formed on IAA‐treated explants with or without putrescine. An increasing number of meristemoids was observed in IAA+BA and BA treatments during culture; in both cases this number was enhanced by the presence of exogenous putrescine. Protease activity was evaluated spectrophotometrically using two synthetic substrates, azocasein and N‐benzoyl‐DL‐arginine‐p‐nitroanilide (BAPNA). In the former, maximum protease activity was observed in IAA+BA‐ and BA‐treated explants on days 10 and 15, respectively, while with IAA activity was lowest, the maximum occurring on days 5–10. In this case exogenous putrescine enhanced protease activity in the presence of IAA alone or with BA, while it decreased it in the presence of BA. BAPNA‐mediated proteolytic activity (serine‐proteases) was highest in IAA+BA‐treated explants, intermediate in BA‐ and not different from controls in IAA‐treated explants. Putrescine only affected proteolytic activity in IAA+BA treatments. The use of specific inhibitors of protease activities indicated that these enzymes belong to two main classes of proteases, that is serine‐ and thiol‐proteases. The pattern of proteolytic activities during culture appeared to be related to the differentiation of meristemoids into vegetative primordia. The effect of exogenous putrescine on protease activity was different depending on different synthetic substrates, developmental patterns, pH and ionic strength.

Antiauxin enhanced microshoot initiation and plant regeneration from epicotyl-originated thin-layer explants of sugarbeet (Beta vulgaris L.)

An in vitro method was developed for microshoot initiation from thin-layer explants prepared from the elongated epicotyls of sugarbeet (Beta vulgaris L.). Intact epicotyls of 14-day-old seedlings were excised from the hypocotyls above the cotyledons and allowed to elongate on De Greef and Jacobs (1979) medium supplemented with 0.2 mg/l 6-benzyladenine, 0.2 mg/l gibberellic acid and 0.1 mg/l indole-3-acetic acid in darkness. After a 21-day-incubation, the elongated epicotyls were halved to obtain apical and basal segments prior to removing the leaves and lateral buds. Subsequently, 5-8 mm long, 2-3 mm wide and 0.8-1.0 mm thick tangential sections were prepared longitudinally from the exterior parts of the halved epicotyls. These thin-layer explants were incubated on microshoot initiating media containing various growth regulators. The combination of 1.0 mg/l 6-benzyladenine and the antiauxin 2,3,5-triiodobenzoic acid (1.0 mg/l) resulted in maximum microshoot development (6.3±0.2 microshoots/thin-layer explant). The final efficiency of our tissue culture system was significantly increased by the NaCl (100 mg/l) initiated in vitro rooting of microshoot originated plantlets.

Formation in vitro of ripe tomato fruits from thin layer explants of flower pedicels

Thin longitudinal sections cut from pedicels of fifteen cultivars of tomato (Lycopersicon esculentum) were grown in vitro on Murashige-Skoog medium supplemented with various concentrations of different auxins and cytokinins. Isatin (an auxin precursor slowly converted to an active auxin) was the most effective source of auxin for the formation of buds without prior root formation, while zeatin was the most effective cytokinin for growth and development of the buds. Flower buds and ripe fruits developed consistently from explants of the cultivar Pixie Hybrid II treated with 10 μM isatin plus 3 μM zeatin as the cytokinin. Fruits developed parthenocarpically, grew to a diameter of about 15 mm, ripened promptly, and possessed normal color and flavor.

Isolation and characterization of mRNAs accumulated during in-vitro flower bud formation.

The development of vegetative and generative buds on thin-layer explants of tobacco (Nicotiana tabacum L. cv. Samsun) has been studied at the level of translatable mRNA to detect changes in the mRNA population during bud initiation and differentiation, and several quantitative differences were found. By differential screening of a cDNA library obtained from flower-bud-regenerating explants we have isolated a group of six cDNA clones representing genes that are preferentially expressed during in-vitro flower bud formation. Nucleotide sequence analysis of one of these cDNAs, pAP8, showed that the most likely open reading frame has some typical characteristics of, and homology with, extensin-like genes. Northern blot analysis and in-situ hybridization suggest a specific role for these extensin-like genes in flower bud initiation on tobacco pedicel explants.

Isatin as an auxin source favoring floral and vegetative shoot regeneration from calli produced by thin layer explants of tomato pedicel.

Thin layer explants taken from the pedicels and peduncles of flowering tomato plants yielded calli with great organogenetic potential. Of the 15 cultivars tested, 7 regenerated roots, shoots and eventually entire fruit-bearing plants. Calli grown on modified Murashige-Skoog medium responded to varied auxins and cytokinins with different morphogenetic patterns. Thus, naphthaleneacetic acid yielded root-producing calli, while the auxin precursor isatin (indole 2,3-dione) caused the production of calli with vegetative and floral shoots, rarely yielding roots. This may be related to isatin's slow, steady conversion to an active auxin (Plant Physiol 41:1485-1488, 1966) in contrast with naphthaleneacetic acid's immediate presentation of a high level of active auxin. The highest incidence of vegetative shoot (100%) and flower (50%) formation was obtained with 10 micromoles isatin and 3 micromoles zeatin. A few of the flowers developed into ripe fruits. The high frequency of induction of vegetative shoots and flowers before roots with isatin suggests its utility in micropropagation from plant tissue cultures.

Regulation of Rhizogenesis by Polyamines in Tobacco Thin Layers

Summary Tobacco thin layer explants were cultured on a rhizogenic medium for 21 days in the presence or absence of methylglyoxal-bis(guanylhydrazone) (MGBG) or cyclohexylamine (CHA), competitive inhibitors of spermidine synthesis through S-adenosylmethionine decarboxylase and spermidine synthase, respectively. On day 21, explants were transferred to hormone-free medium of the same composition with or without MGBG or CHA, alone or in combination with labelled and unlabelled spermidine. The effects of these treatments on free and bound polyamine levels, on the putrescine biosynthetic activity (ornithine decarboxylase, ODC, and arginine decarboxylase, ADC), on labelled spermidine incorporation and on the rhizogenic response were studied. In MGBG-treated explants rhizogenesis was strongly inhibited while with CHA it was less affected. In the former, the addition of spermidine caused a significant reversion of the rooting inhibition (considering both rooting percentage and the mean number of roots per explant), while not in the latter. MGBG induced strong putrescine and spermidine depletion both in the free and bound forms; CHA induced strong spermidine depletion (especially on day 21), but putrescine accumulation. In both treatments, in the presence of labelled spermidine, labelled free and bound putrescine were also detected. A general enhancement in ADC activity was observed in inhibitor-treated explants; the addition of spermidine to the inhibitors caused a further strong increase in ADC activity. These results are discussed in relation to the different metabolic pathways affected by the inhibitors.

Flower-bud formation in explants of photoperiodic and day-neutral Nicotiana biotypes and its bearing on the regulation of flower formation.

The capacity to form flower buds in thin-layer explants was studied in flowering plants of several species, cultivars, and lines of Nicotiana differing in their response to photoperiod. This capacity was found in all biotypes examined and could extend into sepals and corolla. It varied greatly, depending on genotype, source tissue and its developmental stage, and composition of the culture medium, particularly the levels of glucose, auxin, and cytokinin. It was greatest in the two day-neutral plants examined, Samsun tobacco and Nicotiana rustica, where it extended from the inflorescence region down the vegetative stem, in a basipetally decreasing gradient; it was least in the two qualitative photoperiodic plants studied, the long-day plant Nicotiana silvestris and the short-day plant Maryland Mammoth tobacco, the quantitative long-day plant Nicotiana alata and the quantitative short-day plant Nicotiana otophora line 38-G-81, where it was limited to the pedicels (and, in some cases, the sepals). Regardless of the photoperiodic response of the source plants, the response was the same in explants cultured under long and short days. The finding that capacity to form flower buds in explants is present in all Nicotiana biotypes studied supports the idea that it is regulated by the same mechanism(s), regardless of the plant's photoperiodic character. However, the source plants were all in the flowering stage, and no flower-bud formation can be obtained in explants from strictly vegetative Nicotiana plants. Hence, flower formation in the explants is not identical with de novo flower formation in a hitherto vegetative plant: it is rather the expression of a floral state already established in the plant, although it can vary widely in extent and spatial distribution. Culture conditions that permit flower-bud formation in an explant are conditions that maintain the floral state and encourage its expression; conditions under which no flower buds are formed reduce this state and/or prevent its expression.

High-frequency direct somatic embryogenesis in thin layer cultures of hybrid seed geranium (Pelargonium ×hortorum)

Thin layer explants from the hypocotyls of young seedlings of geranium (Pelargonium ×hortorum Bailey) produced somatic embryos in response to thidiazuron (TDZ) or a combination of indoleacetic acid and N6-benzylaminopurine, but the number of embryos was much less in the latter than with TDZ. The development of somatic embryos in cultures of thin layers was rapid and the number of embryos was about eightfold higher than in cultures of whole hypocotyl explants. Thin layer explants of 1-week-old seedlings produced a high number of somatic embryos than those obtained from older seedlings. The cultivar Scarlet Orbit Improved showed better regeneration potential than 'Sprinter Scarlet'. There were no visible differences in total number of somatic embryos produced in the medium containing sucrose or glucose. The addition of glutamine to the induction medium further improved the number of somatic embryos that developed. Regenerated somatic embryos developed into complete plantlets within 6 to 8 weeks of culture initiation. Key words: Pelargonium ×hortorum, geranium, somatic embryogenesis, thin layers, thidiazuron.

The role of hormones on morphogenesis of thin layer explants from normal and transgenic tobacco plants

The role of hormones on morphogenesis of thin layer explants from normal and transgenic tobacco plants

The role of hormones on morphogenesis of thin layer explants from normal and transgenic tobacco plants

The organogenic potential of thin layer stem explants of non‐reproductive tobacco plants was tested on a hormone‐free medium and under various hormonal conditions. A comparison was made between thin layers excised from normal and transgenic plants at the same developmental stage. The transgenic plants were transformed by insertion of TR‐ and TL‐DNA from Agrobacterium rhizogenes 1855 root‐inducing plasmid. The aim was to identify hormonal conditions capable of stimulating the expression of the flowering competence present in the differentiated stem tissues at the induced stage before any visible sign of transition to reproductive development. Flower neoformation, observed at the end of the culture period (day 25), occurred on untransformed thin layers only with kinetin treatment. Explants from transgenic plants showed flower bud regeneration on hormone‐free medium, indoleacetic acid alone (1 μM), kinetin alone (1 μM), and most abundantly on indoleacetic acid plus kinetin (1 μM each). No flower formation was observed on indolebutyric acid plus kinetin (10 μM and 0.1 μM, respectively) in both normal and transgenic explants. The latter treatment enhanced rooting instead, above all in the transgenic explants. On hormone‐free medium vegetative bud formation was well expressed both by untransformed and transgenic explants, and enhanced by the combined, equimolar concentrations of indoleacetic acid and kinetin.The results show that cytokinin allows flowering in florally determined stem explants from normal plants. In the transgenic explants, the flowering response increases when indoleacetic acid is added to cytokinin, thus suggesting a role for auxin in enhancing the expression of the florally determined state in thin cell layers of non‐reproductive plants.

Further experiments on spermidine-mediated floral-bud formation in thin-layer explants of Wisconsin 38 tobacco

We studied the effects of various polyamines on bud regeneration in thin-layer tissue explants of vegetative and floweringNicotiana tabacum L. cv. Wisconsin 38, in which application of exogenous spermidine (Spd) to vegetative cultures causes the initiation and development of some flower buds (Kaur-Sawhney et al. 1988 Planta173, 282). We now show that this effect is dependent on the time and duration of application, Spd being required from the start of the cultures for about three weeks. Neither putrescine nor spermine is effective in the concentration range tested. Spermidine cannot replace kinetin (N(6)-furfurylaminopurine) in cultures at the time of floral bud formation, but once the buds are initiated in the presence of kinetin, addition of Spd to the medium greatly increases the number of floral buds that develop into normal flowers. Addition of Spd to similar cultures derived from young, non-flowering plants did not cause the appearance of floral buds but rather induced a profusion of vegetative buds. These results indicate a morphogenetic role of Spd in bud differentiation.

Spermidine and flower-bud differentiation in thin-layer explants of tobacco

Three lines of evidence indicate a connection between high spermidine levels and floral initiation in thin-layer tissue cultures of Wisconsin-38 tobacco (Nicotiana tabacum L.). (1) Spermidine levels are much higher in floral buds than in vegetative buds. (2) Inhibition of spermidine synthesis by cyclohexylamine prevents the rise in spermidine titer, inhibits floral initiation and promotes the formation of vegetative buds instead. (3) Application of exogenous spermidine causes floral initiation in cultures which would otherwise form vegetative buds.