High-frequency plant regeneration via adventitious shoot formation from deembryonated cotyledon explants of Sesamum indicum L.

Sesamum indicum L. was used as an important oil crop in the world. An efficient protocol for in vitro plant regeneration via adventitious shoot formation from deembryonated cotyledon explants isolated from mature seeds of sesame is developed. Optimal medium for direct adventitious shoot formation was Murashige and Skoog (MS) medium with 22.2 μM 6-benzylaminopurin (BA) and 5.7 μM indole-3-acetic acid (IAA). Abscisic acid (3.8 μM ABA) and AgNO₃ (29.4 μM) were effective in enhancing the frequency of adventitious shoot formation. Preculture of cotyledon explants on high sucrose concentration (6–9%) for 2 wk and subsequent transfer to 3% sucrose enhanced the frequency of adventitious shoot induction. Root formation from the adventitious shoots was easily achieved on MS medium containing 2.7 μM of α-naphthalene acetic acid (NAA). Regenerated plantlets were acclimatized on sand and peat moss (1:1), showing 95% survival with subsequent flowering and seed set. We established the high-frequency plant regeneration via adventitious shoot formation in S. indicum L.     

Hydrogen peroxide acts as a signal molecule in the adventitious root formation of mung bean seedlings

Hydrogen peroxide (H₂O₂), an active oxygen species, is widely generated in many biological systems and mediates various physiological and biochemical processes in plants. In this study we demonstrated that the exogenous H₂O₂ was able to promote the formation and development of adventitious roots in mung bean seedlings. Treatments with 1–100 mM H₂O₂ for 8–18 h significantly induced the formation and development of adventitious roots. Catalase (CAT) and ascorbic acid, which are H₂O₂ scavengers or inhibitors, eliminated the adventitious root-promoting effects of exogenous H₂O₂. H₂O₂ may have a downstream signaling function in the auxin signaling pathway and be involved in auxin-induced adventitious root formation. 2,3,5-Triiodobenzoic acid (TIBA), an inhibitor of auxin polar transport, strongly inhibited adventitious rooting of mung bean seedlings; however, the inhibiting effects of TIBA on adventitious rooting can be partially reversed by the exogenous IBA or H₂O₂. Diphenylene iodonium (DPI) strongly inhibits the activity of NADPH oxidase, which is one of the main sources of H₂O₂ formation in plant cells. DPI treatment strongly inhibited the formation of adventitious roots in mung bean, but the inhibitory effects of DPI on rooting can be partially reversed by the exogenous H₂O₂ or IBA. This indicates that the formation of adventitious roots was blocked once the generation of H₂O₂ through NADPH oxidase was inhibited, and H₂O₂ mediated the IBA-induced adventitious root formation. Furthermore, a rapid increase in the endogenous level of H₂O₂ was detected during incubation with water 12–36 h after the primary root removal in mung bean seedlings. Three hours after the primary root removal, the generation of endogenous H₂O₂ was markedly induced in IBA-treated seedlings in comparison with water-treated seedlings. This implies that IBA induced overproduction of H₂O₂ in mung bean seedlings, and that IBA promoted adventitious root formation via a pathway involving H₂O₂. Results obtained suggest that H₂O₂ may function as a signaling molecule involved in the formation and development of adventitious roots in mung bean seedlings.     

IBA-induced changes in antioxidant enzymes during adventitious rooting in mung bean seedlings: The role of H2O2

The changes in antioxidant enzyme activity during the induction of adventitious roots in mung bean seedlings treated with Indole-3-butyric acid (IBA), hydrogen peroxide (H₂O₂), ascorbic acid (ASA) and diphenylene iodonium (DPI) were investigated. As compared with the controls, treatments of seedlings with 10 μM IBA significantly decreased POD activity by 55% and 49.6% at 3 h and 12 h of incubation, respectively, and significantly increased by 49.8% at 36 h of incubation; treatments of seedlings with 10 mM H₂O₂ significantly decreased POD activity by 42%, 60%, 39% and 38% at 3 h, 12 h, 24 h and 48 h of incubation, respectively, the changes in POD activity were coincident with those in IBA-treated seedlings during the 0–12 h incubation period; treatments of seedlings with 2 mM ASA significantly decreased APX activities by 27% only at 3 h of incubation, the varying trend of POD activity was similar to incubation with water; 10 μM DPI treatments significantly decreased POD activity by 42%, 40%, 54% and 28% at 3 h, 6 h, 12 h and 48 h of treatment, respectively. CAT activities remained at relatively stable levels and no major changes occurred from 0 h to 48 h during the incubation phase of adventitious rooting. The results may imply that CAT, an H₂O₂-metabolizing enzyme, is inactivated by H₂O₂ during the formation of adventitious roots. As compared with the controls, IBA treatments significantly decreased APX activities by 48%, 53% and 66% at 3 h, 9 h and 12 h of treatment, respectively; H₂O₂ treatments significantly decreased APX activities by 59%, 51% and 57% at 3 h, 12 h and 36 h of incubation, respectively; ASA treatments significantly decreased APX activities by 37% only at 3 h of incubation; DPI treatments significantly decreased APX activities by 54%, 53% and 63% at 3 h, 6 h and 12 h of incubation, respectively, and significantly increased APX activity by 106% at 24 h. These results indicated that the influence of IBA, H₂O₂, ASA and DPI on the changes in APX activity were the same as on the changes in POD activity. Furthermore, similar trends in the changes of APX activity and POD activity were observed during the induction and initiation rooting phase. This finding implies that APX and POD serve the same functions, possibly related to the level of H₂O₂, during the formation of adventitious roots. The early decrease of POD and APX activities in the initiation phase of IBA- and H₂O₂-treated seedlings may be one mechanism underlying the IBA- and H₂O₂-mediated facilitation of adventitious rooting.     

Regeneration of salvia (<Emphasis Type="Italic">Salvia officinalis</Emphasis> L.) via induction of meristematic callus

Nodular meristematic callus was induced on the basal cut surface of apical shoot explants of salvia cultured on Murashige and Skoog (MS) medium supplemented with 4.5, 13.5, or 22.5 μM thidiazuron (TDZ). Cultures were incubated in the dark for 1 wk and then transferred to light conditions for 4 wk. A higher percentage of explants developing callus was observed on medium containing either 4.5 or 13.5 μM TDZ, although explants on 4.5 μM developed larger calluses. The callus was maintained on medium containing 4.5 μM TDZ and 0.45 mM ascorbic acid. Shoot differentiation, after each of three successive maintenance passages, was induced from callus grown on medium containing either 4.4 or 8.8 μM benzyladenine (BA). A greater number of shoots were harvested from callus differentiated on BA (4.4 or 8.8 μM) medium with 0.45 mM ascorbic acid added. Shoots developed roots on MS medium supplemented with 4.9 μM of indole-3-butyric acid. The addition of ascorbic acid to the shoot differentiation medium enhanced rooting, number of roots per shoot, and survival rate. Approximately 75% in vitro plantlets were acclimatized to ex vitro conditions. Histological investigations confirmed both adventitious meristem initiation during the callus induction phase, and subsequent organogenic shoot development on the differentiation medium. The novel protocol for the meristematic callus induction and plant regeneration in this study may be useful for biotechnological applications for salvia improvement via genetic transformation or mutagenesis and in vitro propagation approaches.     

Role and relationship of nitric oxide and hydrogen peroxide in adventitious root development of marigold

Several lines of evidence suggest that nitric oxide (NO) and hydrogen peroxide (H₂O₂) are important signal molecules involved in plant development and other physiological processes. Marigold (Tagetes erecta L. ‘Marvel’) was used to understand the role and relationship of NO and H₂O₂ in adventitious root development of plants. The results showed that the effects of H₂O₂ or NO on adventitious root organogenesis of explants were dose dependent, with maximal biological responses at 200 μM H₂O₂ or 50 μM NO donor sodium nitroprusside (SNP). The results also indicated the importance of both putative NO synthase (NOS)-like and nitrate reductase (NR) enzymes, which might be responsible for the production of NO in explants during rooting. Additionally, guanosine 3′, 5′ -cyclic monophosphate (cGMP) was involved in NO- induced root formation of marigold, but it was not involved in H₂O₂- mediated rooting process. The root number and length of explants treated with NO and H₂O₂ simultaneously were significantly higher than those of explants treated with H₂O₂ or NO alone. Moreover, NO treatments enhanced endogenous H₂O₂ levels in hypocotyls. Together, these results indicate that NO and H₂O₂ play crucial roles in the adventitious root development of marigold explants both synergistically and independently.     

Shoot organogenesis from petiole explants in the aquatic plant Nymphoides indica

A protocol for rapid shoot organogenesis from petiole explants of the ornamental aquatic plantNymphoides indica L. Thwaites O. Kuntze was developed for use in future mutation breeding and cultivar selection studies. Optimum culture conditions for shoot organogenesis were determined. Effects of factorial combinations of 2-iP, BA or kinetin (0–25 μM) in factorial combination with IAA or NAA (0–25 μM) were examined. On the basis of regeneration frequency (80%) and adventitious shoot number (11.5 shoots per explant), most efficient shoot organogenesis occurred on petiole explants cultured on a basal medium consisting of full-strength MS inorganic salts, 0.56 mM myo-inositol, 1.2 μM thiamine-HCl, 116.8 mM sucrose supplemented with 10 μM BA and 20 μM IAA and solidified with 0.8% TC agar. Formation of adventitious shoots by direct and indirect shoot organogenesis from the same explant was verified by histological sectioning. With the exception of variegated leaf production on a single adventitious shoot produced in the presence of 25 μM kinetin and 15 μM NAA, no visible phenotypic abnormalities were observedin vitro in any of the shoots generated. Solid achlorophyllous adventitious shoots were recovered following culture of this variegated leaf tissue. Plantlets were easily acclimatized toex vitro conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

<Emphasis Type="Italic">In vitro</Emphasis> adventitious shoot regeneration of the medicinal plant meadowsweet (<Emphasis Type="Italic">Filipendula ulmaria</Emphasis> (L.) Maxim)

Filipendula ulmaria (L.) Maxim (meadowsweet) is a medicinal plant that is claimed to have several biological activities, including anti-tumor, anti-carcinogenic, anti-oxidant, anti-coagulant, anti-ulcerogenic, anti-microbial, anti-arthritic, and immunomodulatory properties. This report describes, for the first time, an efficient plant regeneration system for F. ulmaria via adventitious shoot development from leaf, petiole, and root explants cultured on Murashige and Skoog’s minimal organics medium containing different concentrations of thidiazuron (TDZ), benzyladenine, and kinetin either alone or in combination with different auxins. Relatively extensive/prolific shoot regeneration was observed in all three explant types with TDZ in combination with indole-3-acetic acid (IAA). Gibberellic acid (GA₃), TDZ, and IAA combinations were also tested. The best shoot proliferation was observed among root explants cultured on media supplemented with 0.45 μM TDZ + 2.85 μM IAA + 1.44 μM GA₃. Regenerated shoots were transferred to rooting media containing different concentrations of either IAA, indole-3-butyric acid (IBA), naphthalene acetic acid, or 2,4-dichlorophenoxyacetic acid. Most shoots developed roots on medium with 2.46 μM IBA. Rooted explants were transferred to vermiculite in Magenta containers for a 2-wk acclimatization period and then finally to plastic pots containing potting soil. The plantlets in soil were kept in growth chambers for 2 wk before transferring to greenhouse conditions.     

Pretreatment in thidiazuron improves the in vitro shoot induction from leaves in Curculigo orchioides Gaertn., an endangered medicinal plant

Leaf regeneration via direct induction of adventitious shoots obtained from an endangered medicinal plant, Curculigo orchioides Gaertn. by pretreating with thidiazuron. C. orchioides is an endangered medicinal herb belonging to the family Hypoxidaceae. Direct inoculation of leaf pieces on MS medium supplemented with various concentrations of BAP (2–8 μM) or TDZ (2–8 μM) alone or in combination with NAA (0.5 and 1.0 μM) produced low shoot induction both in terms of % response and number of shoots per explant. Hence, leaf explants were pretreated with 15, 25 or 50 μM thidiazuron (TDZ), for 6, 24 or 48 h with the aim of improving shoot regeneration from cultured explants. After pretreatment, explants were transferred to an agar solidified MS medium that was supplemented with BAP (4 μM), TDZ (6 μM), BAP (4 μM) + NAA (1.0 μM), TDZ (6 μM) + NAA (0.5 μM). Control explants were incubated directly on the medium without any pretreatment. The pretreatment of explants with 15 μM TDZ for 24 h significantly promoted the formation of adventitious shoots and the maximum response was observed on MS medium supplemented with 6 μM TDZ. In this medium, 96 % cultures responded with an average number of 16.2 adventitious shoots per explant. The percentage of leaf explants producing shoots and the average number of shoots per explant were significantly improved when TDZ pretreated leaves were cultured onto MS medium supplemented with BAP or TDZ alone or in combination with NAA. The rooted plantlets were successfully transplanted to soil with 90% success. The present investigation indicated the stimulatory role of TDZ pretreatment in regulating shoot regeneration from leaf explants of C. orchioides.     

Function of nitric oxide and superoxide anion in the adventitious root development and antioxidant defence in <Emphasis Type="Italic">Panax ginseng</Emphasis>

The involvement of NO in O₂ ^·− generation, rootlet development and antioxidant defence were investigated in the adventitious root cultures of mountain ginseng. Treatments of NO producers (SNP, sodium nitroprusside; SNAP, S-nitroso-N-acetylpenicillamine; and sodium nitrite with ascorbic acid), and NO scavenger (PTIO, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl3-oxide) revealed that NO is involved in the induction of new rootlets. Severe decline in number of new rootlets compared to the control under PTIO treatment indicates that NO acts downstream of auxin action in the process. NO producers (SNP, SNAP and sodium nitrite with ascorbic acid) activated NADPH oxidase activity, resulting in greater O₂ ^·− generation and higher number of new rootlets in the adventitious root explants. Moreover, treatment of diphenyliodonium chloride, a NADPH oxidase inhibitor, individually or along with SNP, inhibited root growth, NADPH oxidase activity and O₂ ^·− anion generation. NO supply also enhanced the activities of antioxidant enzymes that are likely to be responsible for reducing H₂O₂ levels and lipid peroxidation as well as modulation of ascorbate and non-protein thiol concentrations in the adventitious roots. Our results suggest that NO-induced generation of O₂ ^·− by activating NADPH oxidase activity is related to adventitious root formation in mountain ginseng.     

Shoot organogenesis and somatic embryogenesis from leaf and shoot explants of <Emphasis Type="Italic">Ochna integerrima</Emphasis> (Lour)

Ochna integerrima is a medicinal and ornamental plant in Southeastern Asia. It has been listed as a rare and endangered species in China. Here we studied the effects of plant growth regulators and their concentrations on the induction of somatic embryogenesis and shoot organogenesis from leaf and shoot explants of O. integerrima for the first time. Cytokinins played a crucial role in somatic embryogenesis and shoot organogenesis. Among them, a higher concentration of thidiazuron (10.0–15.0 μM TDZ) could induce both somatic embryogenesis and adventitious shoot formation whereas low concentrations of TDZ (5.0 μM) could only induce adventitious shoots. However, 6-benzyladenine (BA at 5–15 μM) could only induce adventitious shoots. Shoot explants induced more adventitious shoots and somatic embryos than leaf explants when cultured on medium with the same concentration (5–15 μM) of TDZ or 15 μM BA. Medium containing 0.5 μM α-naphthaleneacetic acid and 8 μM indole-3-butyric acid and 0.1% activated charcoal could induce adventitious roots within 1 month. An efficient mass propagation and regeneration system has been established.     

The Effect of Different Plant Growth Regulators on Adventitious Shoot Formation from Virginia Pine (Pinus virginiana) Zygotic Embryo Explants

The effects of different plant growth regulators on in vitro adventitious shoot formation in Virginia pine (Pinus virginiana Mill.) zygotic embryo explants were quantitatively evaluated. Using Tang and Ouyang (1999) (TE) basal medium supplemented with 11.4 μM indole-3-acetic acid (IAA) and 2.2 μM N⁶-benzyladenine (BA), callus was observed after 3–6 weeks of culture. Calluses were transferred to TE basal medium supplemented with 0.49 μM indole-3-butyric acid (IBA) and 8.8 μM BA for 6–9 weeks, where they produced numerous small shoot primordia. They were then transferred to TE basal medium supplemented with 0.49 μM IBA and 4.4 μM BA to promote growth and elongation of adventitious shoots. After elongated shoots were transferred to TE medium containing 0.05 μM α-naphthaleneacetic acid (NAA) for 6 weeks, adventitious roots were formed. Regenerated plantlets were established in soil in greenhouse.     

Acetylene reduction and indoleacetic acid production by Azospirillum isolates from Cactaceous plants

Azospirillum isolates were obtained from rhizosphere soil and roots of three cactaceae species growing under arid conditions. All Azospirillum isolates from rhizosphere and roots ofStenocereus pruinosus andStenocereus stellatus were identified asA. brasilense; isolates of surface-sterilized roots fromOpuntia ficus-indica were bothA. brasilense andA. lipoferum. Azospirilla per g of fresh root in the three species ranged from 70×10³ to 11×10³. The most active strains in terms of C₂H₂ reduction (25–49.6 nmol/h·ml) and indoleacetic acid (IAA) production (36.5–77 μg/ml) were those identified asA. brasilense and isolated from Stenocereus roots.A. lipoferum isolated from Opuntia roots produced low amounts of IAA (6.5–17.5 μg/ml) and low C₂H₂-reduction activity (17.8–21.2 nmol/h·ml).     

In vitro plant regeneration from immature cotyledon explants of macadamia (Macadamia tetraphylla L. Johnson)

The macadamia tree, an Australian native, is highly valued for its nuts. Macadamia improvement programs so far have relied on conventional breeding and selection. The production of improved cultivars required to meet future demands could be accelerated by the application of modern biotechnological techniques, but this requires an efficient and reproducible regeneration system that has not yet been established for macadamia. We report here shoot regeneration from immature cotyledon explants of macadamia. Adventitious buds were induced on the cotyledon explants from fruits collected at 140 and 190 days after full bloom (DAFB) on MS medium supplemented with either 10 or 15 μM TDZ. The addition of 2% coconut milk (CM) to 10 μM TDZ containing media resulted in enhanced adventitious bud induction from 190 DAFB explants. Further shoot development from the induced buds was depressed in media containing TDZ + CM; the addition of 0.001 μM IAA to this combination doubled shoot development, from 1.9–3.9 shoots per explant. The transfer of bud clumps to media supplemented with 8.8 μM BA alone or in combination with either 0.14 μM GA₃ or 0.001 μM IAA significantly increased shoot production from the previously induced explants by 1.5–2 times of that observed in TDZ + CM medium. Histological examinations revealed that shoot regeneration was primarily by organogenesis originating from cells on or just below the cut surfaces of explants.     

Arsenic-induced root growth inhibition in mung bean (<Emphasis Type="Italic">Phaseolus aureus</Emphasis> Roxb.) is due to oxidative stress resulting from enhanced lipid peroxidation

Arsenic (As) toxicity and its biochemical effects have been mostly evaluated in ferns and a few higher plants. In this study, we investigated the effect of As (10.0 and 50.0 μM) on seedling growth, root anatomy, lipid peroxidation (malondialdehyde and conjugated dienes), electrolyte leakage, H₂O₂ content, root oxidizability and the activities of antioxidant enzymes in mung bean (Phaseolus aureus Roxb.). Arsenic significantly enhanced lipid peroxidation (by 52% at 50.0 μM As), electrolyte leakage and oxidizability in roots. However, there was no significant change in H₂O₂ content. Arsenic toxicity was associated with an increase in the activities of superoxide dismutase (SOD), guaiacol peroxidase (GPX) and glutathione reductase (GR). In response to 50.0 μM As, the activities of SOD and GR increased by over 60% and 90%, respectively. At 10.0 μM As, the activity of ascorbate peroxidase (APX) increased by 83%, whereas at 50.0 μM it declined significantly. The catalase (CAT) activity, on the other hand, decreased in response to As exposure, and it corresponded to the observed decrease in H₂O₂ content. We conclude that As causes a reduction in root elongation by inducing an oxidative stress that is related to enhanced lipid peroxidation, but not to H₂O₂ accumulation.     

Direct shoot bud induction and plant regeneration in <Emphasis Type="Italic">Capsicum frutescens</Emphasis> Mill.: influence of polyamines and polarity

Direct shoot bud induction and plant regeneration was achieved in Capsicum frutescens var. KTOC. Aseptically grown seedling explants devoid of roots, apical meristem and cotyledons were inoculated in an inverted position in medium comprising of Murashige and Skoog (Physiol Plant 15:472–497, 1962) basal medium supplemented with 2-(N-morpholine) ethanesulphonic acid buffer along with 2.28 μM indole-3-acetic acid, 10 μM silver nitrate and either of 13.31–89.77 μM benzyl adenine (BA), 9.29–23.23 μM kinetin, 0.91–9.12 μM zeatin, 2.46–9.84 μM 2-isopentenyl adenine. Profuse shoot bud induction was observed only in explants grown on a media supplemented with BA (26.63 μM) as a cytokinin source and 19.4 ± 4.2 shoot buds per explant was obtained in inverted mode under continuous light. Incorporation of polyamine inhibitors in the culture medium completely inhibited shoothoot bud induction. Incorporation of exogenous polyamines improved the induction of shoot buds under 24 h photoperiod. These buds were elongated in MS medium containing 2.8 μM gibberellic acid. Transfer of these shoots to hormone-free MS medium resulted in rooting and rooted plants were transferred to fields. This protocol can be efficiently used for mass propagation and presumably also for regeneration of genetically transformed C. frutescens.     

Improved micropropagation in Polygala myrtifolia

Summary Stem segments from apical shoot tips of Polygala myrtifolia were used as primary explants to establish in vitro cultures. Axillary shoots produced on non-contaminated explants were excised and recultured in the same medium to increase the stock of shoot cultures. Equal molar concentrations of five cytokinins [2-isopentenyladenine, kinetin, zeatin, N ⁶-benzyladenine (BA), and adenine] were tested for ability to induce axillary shoot development from double-node stem segments. The highest rate of axillary shoot proliferation was induced on Murashige and Skoog agar medium supplemented with 1.8 μM BA. Seven indole-3-acetic acid (IAA) concentrations (0, 2.9, 5.7, 8.6, 11.4, 14.3, 17.1 μM) were tested to determine the optimum conditions for in vitro rooting of microshoots. Up to 72% of the microshoots rooted with 14.3 μM IAA. Other auxins tested, α-naphthaleneacetic acid and indole-3-butyric acid, were less effective than IAA in inducing adventitious root formation. All rooted plantlets having more than three roots were successfully established in soil.     

In vitro adventitious bud regeneration from internode segments of beech

Internode explants collected from in vitro grown shoots of two clones of Fagus sylvatica L. (European beech) and five clones of F. orientalis Lipski (Oriental beech) were used to evaluate their bud regeneration capacity. Adventitious shoot-buds formed on callus, which developed from internode segments cultured in a Woody Plant Medium supplemented with different concentrations of either thidiazuron (TDZ) or benzyladenine (BA). After 4 weeks of culture on induction media, the explants were transferred to a proliferation medium supplemented with 2.2 μM BA, 9.1 μM zeatin and 2.9 μM indole-3-acetic acid (IAA) for another 8 weeks. Medium containing TDZ was much more efficient than medium containing BA in inducing adventitious buds, the optimal TDZ concentration being 4.5 μM and the optimal BA concentration 17.8 μM. Genotypic variation in shoot regeneration capacity was observed among the two Fagus species and between clones within each species, with a significant interaction between TDZ concentration and genotype regarding mean bud number. Thidiazuron induction medium supplemented with a range of individual auxins was investigated, and it was found that IAA or indole-3-butyric acid at 2.9 μM enhanced the bud forming capacity of explants. Morphogenic response varied significantly with the position of the internode along the stem. The highest regeneration potential was obtained from apical internodes, while those distal to the apex were the least productive. Elongated shoots of adventitious origin can be readily proliferated by axillary branching. This revised version was published online in June 2006 with corrections to the Cover Date.     

Role of polyamines in adventitious shoot morphogenesis from cotyledons of cucumber <Emphasis Type="Italic">in vitro</Emphasis>

The relationship between polyamines (PAs) metabolism and adventitious shoot morphogenesis from cotyledons of cucumber was investigated in vitro. The endogenous levels of free putrescine (Put) and spermidine (Spd) in the explants decreased sharply, whereas endogenous spermine (Spm) increased during adventitious shoot morphogenesis. The presence of 1–15 mM Put, 1–2 mM Spd, 0.05–1 mM Spm, 5–10 M aminoethoxyvinylglycine (AVG) or 5 M AVG together with 50 M 1-aminocyclopropane-1-carboxylic acid (ACC) in the regeneration medium could promote adventitious shoot formation. Conversely, 1–5 mM D-arginine (D-Arg) or 0.01–0.1 mM methylglyoxal bis-guganylhydrazone (MGBG) inhibited regeneration; and 0.005–0.05 mM ACC displayed little or no evident effects. The explants growing on medium containing 5 M AVG produced higher levels of free Put and Spm, and on medium containing 5 mM Put the explants responded similarly to the AVG-treated explants. However, the exogenous use of 1 mM D-Arg reduced the levels of Put, Spd and Spm, and 0.1 mM MGBG reduced the levels of free Spd and Spm. Moreover, although the explants cultured on medium containing Put and MGBG enhanced ethylene production, AVG and D-Arg inhibited ethylene biosynthesis. This study shows the PAs requirement for the formation of adventitious shoot from cotyledons of cucumber in vitro and the enhanced adventitious shoot morphogenesis may be associated with the elevated level of endogenous free Spm, albeit the promotive effect of PAs on adventitious shoot morphogenesis may not be related to ethylene metabolism.     

Effects of sodium nitroprusside on callus induction and shoot regeneration in micropropagated Dioscorea opposita

The effects of sodium nitroprusside (SNP) on callus induction and shoot regeneration of Dioscorea opposite Thunb. have been studied. Application of 40 μM of SNP depresses accumulation of H₂O₂ in tuber explants of Dioscorea opposita markedly. Supplementation of 40 μM of SNP to the Murashige and Skoog medium with combinations of benzylaminopurine (3 mg dm⁻³) and naphthaleneacetic acid (0.5 mg dm⁻³) reduces the browning of explants and increases the frequency of callus induction from tuber explants significantly. The regeneration frequency of adventitious shoot shows a significant increase in the presence of SNP. Further analysis indicates that treatment with 40 μM of SNP results in significant decreases in catalase and peroxidase activity, while increasing the activity of superoxide dismutase. Supplementation with 40 μM of SNP also promotes the accumulation of non-enzymic antioxidants, including proline and glutathione. The effects on callus induction and shoot regeneration promoted by SNP were reversed by the nitric oxide (NO) scavenger 2-(4-carboxyphenyl)- 4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide. These results indicate that the exogenously applied NO-donor SNP alleviates browning of tuber explants by reducing H₂O₂ accumulation, thereby promoting a higher in vitro proliferation frequency of D. opposita.     

Fusaric acid induces apoptosis in saffron root-tip cells: roles of caspase-like activity, cytochrome c, and H2O2

Programmed cell death (PCD), now known as apoptosis, is accompanied by specific morphological features. In this study, fusaric acid, a fusarium mycotoxin, was used to examine cell death in saffron (Crocus sativus Linnaeus) roots, using several apoptosis assays. Our results show that moderate FA doses (50–100 μM) induce apoptotic features while high FA doses (> 200 μM) stimulate necrosis. The apoptotic-like features induced by moderate doses of FA include chromatin condensation, formation of condensed chromatin spheres which bud from the nucleus, fragmentation of nucleosomal DNA into ∼ 180 bp fragments, exposure of phosphatidyl serine to the external membrane leaflet, delivery of cytochrome c to cytosol, and generation of H₂O₂. These apoptotic alterations in root cells are not observed in the presence of serine protease, caspase-1 or caspase-3 inhibitors. It is proposed that production of H₂O₂ and release of cytochrome c into the cytosol may activate caspase-like proteases and thus establish the apoptotic pathway. As nuclei budding spheres formed in plant root cells after exposure to 50–100 μM FA doses seem to be digested inside the cytosol, we suggest labeling them as internal apoptotic bodies (IAB) that may be more informative than previously used term, apoptotic-like bodies.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .