Polyamine and nitric oxide levels relate with morphogenetic evolution in somatic embryogenesis of <Emphasis Type="Italic">Ocotea catharinensis</Emphasis>

In this study we examined the effect of polyamines (PAs) putrescine (Put), spermidine (Spd) and spermine (Spm) on growth, morphology evolution, endogenous PAs levels and nitric oxide (NO) release in Ocotea catharinensis somatic embryo cultures. We observed that Spd and Spm reduced culture growth, permitted embryo morphogenetic evolution from the earliest to last embryo development stages, increased endogenous PAs levels, and induced NO release in O. catharinensis somatic embryos. On the other hand, Put had little effect on these parameters. Spd and Spm could successfully be used to promote somatic embryo maturation in O. catharinensis. The results suggest that Spd and Spm have an important role during the growth, development and morphogenetic evolution of somatic embryos, through alterations in the endogenous nitric oxide and PAs metabolism in this species.     

Effect of root-applied spermidine on growth and respiratory metabolism in roots of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>) seedlings under hypoxia

The effects of exogenous spermidine (Spd) application to hypoxic nutrient solution on the contents of endogenous polyamines (PAs) and respiratory metabolism in the roots of cucumber (Cucumis sativus L.) seedlings were investigated. Cucumber seedlings were grown hydroponically in control and hypoxic nutrient solutions with and without addition of Spd at a concentration of 0.05 mM. The activities of key enzymes involved in the tricarboxylic acid cycle (TCAC), such as succinate dehydrogenase (SDH) and isocitrate dehydrogenase (IDH), were significantly inhibited under root-zone hypoxia with dissolved oxygen (DO) at 1 mg/l. In contrast, the activities of enzymes involved in the process of fermentation, such as pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH), and alanine aminotransferase (AlaAT), were significantly increased. Thus, aerobic respiration was inhibited and fermentation was enhanced in the roots of cucumber seedlings as a result of decreasing ATP content to inhibit the dry weight of seedlings under hypoxic stress. Moreover, the contents of free, soluble conjugated, and insoluble bound putrescine (Put), Spd, and spermine (Spm) in the roots of cucumber seedlings were significantly increased under hypoxia stress. Interestingly, application of Spd to hypoxic roots markedly suppressed the accumulation of free Put and, in contrast, promoted an increase in free Spd and Spm, as well as soluble conjugated and insoluble bound Put, Spd, and Spm contents. From these data, we deduced that exogenous Spd promotes the conversion of free Put into free Spd and Spm, and soluble conjugated and insoluble bound PAs under hypoxia stress. Furthermore, the activities of LDH, PDC, and ADH were suppressed and, in contrast, the activities of SDH and IDH were enhanced by application of exogenous Spd to hypoxic roots. As a result, aerobic respiration was enhanced but fermentation metabolism was inhibited in the roots of cucumber seedlings, leading to an increase in ATP content to alleviate the inhibited dry weight of seedlings due to hypoxia stress. These results suggest that application of Spd to hypoxic nutrient solution promoted conversion of free Put into free Spd and Spm as well as soluble conjugated and insoluble bound PAs, further enhanced IDH and SDH activities, and inhibited ethanol fermentation and lactate fermentation, resulting in increased ATP content and eventually enhanced tolerance of cucumber plants to root-zone hypoxia.     

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.     

Effect of polyamines on in vitro somatic embryogenesis in <Emphasis Type="Italic">Momordica</Emphasis><Emphasis Type="Italic">charantia</Emphasis> L.

The effects of exogenous polyamines (PAs) on enhancement of somatic embryogenic calli was investigated in Momordica charantia L. in vitro. Induction of somatic embryogenesis (SE) in leaf explants of M. charantia after 21 days of culture in Murashige and Skoog (MS) medium was determined using scanning electron microscopy. During induction of SE there were high titers of Putrescine (Put) as compared to Spermidine (Spd) and Spermine (Spm), a prerequisite for cell division. Addition of PAs to the embryogenic media resulted in an increase in fresh weights and number of somatic embryos of 21-day old embryogenic calli. Put at a concentration of 1 mM showed maximum increase in fresh weights of embryogenic calli (5 fold) and number of somatic embryos produced per 0.2 g of callus (2.5 fold). Moreover addition of PAs to the embryogenic media resulted in lowering of endogenous free PA level of 21-day old embryogenic calli. Thus, when the media was supplemented with exogenous PAs a positive correlation was found to exist between Somatic Embryogenesis enhancement and decrease in endogenous free PA levels.     

Efficient regeneration and antioxidative enzyme activities in <Emphasis Type="Italic">Brassica rapa</Emphasis> var. <Emphasis Type="Italic">turnip</Emphasis>

The regeneration potential and antioxidative enzyme activities of economically important Brassica rapa var. turnip were evaluated. Calli were induced from leaf explants of seed-derived plantlets on Murashige and Skoog (MS) medium incorporated with different concentrations of various plant growth regulators (PGRs). The highest leaf explant response (83%) was recorded for 2.0 mg l⁻¹ benzyladenine (BA) and 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA). Subsequent subculturing of callus after 3 weeks of culture, on medium with similar compositions of PGRs, induced shoot organogenesis. The highest shoot induction response (83%) was recorded for 5.0 mg l⁻¹ BA after 5 weeks of transfer. However, 7.8 shoots/explant were recorded for 2.0 mg l⁻¹ BA. The transferring of shoots to elongation medium resulted in 5.1-cm-long shoots on 10 mg l⁻¹ of gibberellic acid (GA₃). Rooted plantlets were obtained on MS medium containing different concentrations of indole butyric acid (IBA). The determination of activities of antioxidative enzymes (superoxide dismutase [SOD], ascorbate peroxidase [APX], catalase [CAT], glutathione peroxidase [GPX], and peroxidase [POD]) revealed involvement of these enzymes in callus formation and differentiation. All of the activities were interlinked with each other and played significant roles in the scavenging of toxic free radicals. This study will help in the advancement of a regeneration protocol for B. rapa var. turnip and the understanding of the functions of antioxidative enzymes in plant differentiation.     

<Emphasis Type="Italic">In vitro</Emphasis> organogenesis and antioxidant enzymes activity in <Emphasis Type="Italic">Acanthophyllum sordidum</Emphasis>

The effect of various hormonal combinations on callus formation and regeneration of shoot and root from leaf derived callus of Acanthophyllum sordidum Bunge ex Boiss. has been studied. Proteins and activity of antioxidant enzymes were also evaluated during shoot and root organogenesis from callus. Calli were induced from leaf explants excised from 30-d-old seedlings grown on Murashige and Skoog medium containing 4.52 μM 2,4-dichlorophenoxyacetic acid + 4.65 μM kinetin. Maximum growth of calli and the most efficient regeneration of shoots and roots occurred with 2.69 μM 1-naphthalene acetic acid (NAA), 2.69 μM NAA + 4.54 μM thidiazuron and 2.46 μM indole-3-butyric acid. Protein content decreased in calli and increased significantly during regeneration of shoots from callus. Superoxide dismutase activity decreased in calli comparing to that of seedlings, then increased in regenerated shoots and roots. High catalase activity was detected in seedlings and regenerated shoots, whereas high peroxidase activity was observed in calli and regenerated roots.     

Effect of salt stress on physiological and antioxidative responses in two species of <Emphasis Type="Italic">Salicornia</Emphasis> (<Emphasis Type="Italic">S. persica</Emphasis> and <Emphasis Type="Italic">S. europaea</Emphasis>)

The effects of salt stress on growth parameters, free proline content, ion accumulation, lipid peroxidation, and several antioxidative enzymes activities were investigated in S. persica and S. europaea. The seedlings were grown for 2 months in half-strength Hoagland solution and treated with different concentrations of NaCl (0, 85, 170, 340, and 510 mM) for 21 days. The fresh and dry weights of both species increased significantly at 85 and 170 mM NaCl and decreased at higher concentrations. Salinity increased proline content in both the species as compared to that of control. Sodium (Na⁺) content in roots and shoots increased, whereas K⁺ and P_i content in both organs decreased. At all NaCl concentrations, the total amounts of Na⁺ and K⁺ were higher in shoots than in roots. Malondialdehyde (MDA) content declined at moderate NaCl concentrations (85 and 170 mM) and increased at higher levels. With increased salinity, superoxide dismutase (SOD), catalase (CAT), and guaiacol peroxidase (GPX) activities also increased gradually in both species. In addition, it seems that GPX, CAT, and SOD activities play an essential protective role in the scavenging reactive oxygen species (ROS) in both species. Native polyacrylamide gel electrophoresis (PAGE) indicated different isoform profiles between S. persica and S. europaea concerning antioxidant enzymes. These results showed that S. persica exhibits a better protection mechanism against oxidative damage and it is more salt-tolerant than S. europaea possibly by maintaining and/or increasing growth parameters, ion accumulation, and antioxidant enzyme activities.     

In vitro regeneration of <Emphasis Type="Italic">Carlina acaulis</Emphasis> subsp. <Emphasis Type="Italic">simplex</Emphasis> from seedling explants

The aim of the study was to obtain an efficient system for Carlina acaulis subsp. simplex propagation. The experimental materials were shoot tips, fragments of hipocotyls, cotyledons and roots isolated from 10-day-old seedlings. The explants were transferred to the proliferation medium supplemented with different types of cytokinin: BA (13.3 μM), kinetin (13.9 μM) and zeatin (13.7 μM) in combination with NAA (0.54 μM). The best morphogenetic response was observed when explants were cultured on the BA supplemented medium. The maximum shoot organogenesis frequency was observed for shoot tip (nearly 94%). On average 8.6 axillary shoots were induced per explant. Multiplication rate increased during the first three subcultures. The shoots revealed a wide range of morphogenetic responses. Differences were observed in the presence or absence of hair on the surface of lamina. These changes had epigenetic character and were the effect of changes in DNA methylation, which is shown by differences in methylation pattern between 18S rRNA and 25S rRNA genes in the analyzed regenerated plants. Nearly 94% of plantlets were rooted on auxin lacking medium. Addition of auxin (NAA or IAA) increased both the rooting percentage (100%) and the number of roots per shoot, but their growth was inhibited. Shortening of the auxin exposition time reduced the number of roots. Moreover, high efficiency (90%) was observed for ex vitro rooting. Plantlets with a large number of roots survived better than the ones with only a few roots. Plants were able to flower and gave viable seeds.     

Large-scale somatic embryogenesis and regeneration of <Emphasis Type="Italic">Panax notoginseng</Emphasis>

An efficient system for inducing somatic embryogenesis in Panax notoginseng was established using shaker flasks and bioreactor cultures; furthermore, regenerated plantlets were successfully transferred to ex vitro soil conditions. Embryogenic callus was induced from segments of adventitious roots incubated on Murashige and Skoog (MS) medium containing 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) after 5 weeks of culturing. The highest frequency (100%) of somatic embryogenesis, with a mean of 32.7 somatic embryos per callus, was obtained on embryogenic callus incubated on a medium containing 0.5 mg/L 2,4-D. To scale-up somatic embryo formation, 10 g (~1.65 × 10⁴) of early globular-stage somatic embryos were incubated in a 3 L airlift bioreactor containing 1.5 L 1/2 MS medium without plant growth regulators (PGRs) for a period of 4 weeks; these globular-stage somatic embryos then developed into cotyledonary embryos. When maintained on PGR-free medium, the cotyledonary embryos developed roots but did not develop shoots. However, when they were treated with gibberellic acid (GA₃), they continued to germinate and transformed into plantlets after 2 weeks of culture. Plantlets with well-developed shoots and roots were transferred to an autoclaved vermiculite and perlite mixture, acclimatized for a period of 3 months and successfully transferred to forest mountain soil. Following overwintering, these plants produced new growth.     

Exogenous spermidine alleviates fruit granulation in a <Emphasis Type="Italic">Citrus</Emphasis> cultivar (<Emphasis Type="Italic">Huangguogan</Emphasis>) through the antioxidant pathway

The role of exogenous spermidine (Spd) in alleviating fruit granulation in the grafted seedlings of a Citrus cultivar (Huangguogan) was investigated. Granulation resulted in increased electrical conductivity, cell membrane permeability, and total pectin, soluble pectin, cellulose, and lignin contents. However, it decreased the activities of superoxide dismutase, peroxidase, and catalase, as well as the (Spd + Spm):Put ratio. The application of exogenous Spd onto Huangguogan seedlings significantly increased proline and ascorbate contents, but decreased the H₂O₂ and O ₂ ^?· levels, which suggested that exogenous Spd provided some protection from oxidative damage. In addition, exogenous Spd decreased cell membrane permeability and MDA content, and increased the (Spd + Spm):Put ratio. The activities of antioxidant enzymes, such as catalase, peroxidase, and superoxide dismutase, were increased in Spd-treated seedlings affected by fruit granulation, resulting in a decrease in oxidative stress levels. The protective effects of Spd were reflected by a decrease in superoxide levels through osmoregulation, increased proline and ascorbate contents, and increased antioxidant activities. Our observations reveal the importance of exogenous Spd in alleviating citrus fruit granulation.     

Exogenous spermidine and spermine enhance cadmium tolerance of <Emphasis Type="Italic">Potamogeton malaianus</Emphasis>

In order to investigate the effects of spermidine (Spd) and spermine (Spm) on cadmium stress, the content of chlorophyll, hydrogen peroxide (H₂O₂), malondialdehyde (MDA), soluble protein and proline, the rate of O₂^·− generation, and activities of antioxidant enzymes (superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), and glutathione reductase (GR)) in Potamogeton malaianus Miq. were measured. Exogenous application of Spd or Spm significantly enhanced the level of proline, retarded the loss of chlorophyll, enhanced photosynthesis, decreased the rate of O₂^·− generation and H₂O₂ content, and prevented Cd-induced lipid peroxidation. Spd and Spm also effectively maintained the balance of antioxidant enzyme activities under Cd stress; however, GR activity was found to increase only slightly in response to polyamines (PAs). The antioxidant systems, which were modified by PAs, were able to moderate the radical-scavenging system and to lessen in this way the oxidative stress. These results suggest that both Spd and Spm can enhance Cd tolerance of P. malaianus.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of medicinal plant <Emphasis Type="Italic">Centella asiatica</Emphasis>

This paper describes multiple shoot regeneration from leaf and nodal segments of a medicinally important herb Centella asiatica L. on Murashige and Skoog’s (MS) medium supplemented with a range of growth regulators. The highest number of multiple shoots was observed on MS augmented with 3.0 mg dm⁻³ N⁶-benzylaminopurine (BAP) and 0.05 mg dm⁻³ α-naphthaleneacetic acid (NAA). Leaf explant showed maximum percentage of cultures regenerating shoots (81.6 %), with the highest shoot number (8.3 shoots per explant) and the shoot length (2.1 cm) whereas, nodal explant showed less number of shoots with callus formation at the base cut end. Successive shoot cultures were established by repeatedly sub-culturing the original explant on a fresh medium. Rooting of in vitro raised shoots was best induced on half strength MS supplemented with 0.5 mg dm⁻³ indole-3-butyric acid (IBA) with highest percentage of shoot regenerating roots (76.8 %) with 3–4 roots per shoot. Plantlets were acclimated in Vermi-compost and eventually established in soil. Contents of chlorophyll, total sugars, reducing sugars and proteins were estimated in leaf tissue from both in vivo and in vitro raised plants. Chlorophyll content was higher in in vivo plants, whereas other three components were higher in in vitro plants.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of northern red oak (<Emphasis Type="Italic">Quercus rubra</Emphasis> L.)

In vitro propagation of northern red oak (Quercus rubra) shoots was successful from cotyledonary node explants excised from 8-wk-old in vitro grown seedlings. Initially, four shoots per explant were obtained on Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzylaminopurine (BA), 0.45 μM thidiazuron (TDZ), and 500 mg l⁻¹ casein hydrolysate (CH) with a regeneration frequency of 64.7% after 3 wk. Subculturing explants (after harvesting shoots) to fresh treatment medium significantly increased shoot bud regeneration (16.6 buds per explant), but the buds failed to develop into shoots. A higher percentage (73.3%) of the explants regenerated four shoots per explant on woody plant medium (WPM) supplemented with 4.4 μM BA, 0.29 μM gibberellic acid (GA₃), and 500 mg l⁻¹ CH after 3 wk. Explants subcultured to fresh treatment medium after harvesting shoots significantly increased shoot regeneration (16 shoots per explant). Shoot elongation was achieved (4 cm) when shoots were excised and cultured on WPM supplemented with 0.44 μM BA and 0.29 μM GA₃. In vitro regenerated shoots were rooted on WPM supplemented with 4.9 μM indole-3-butyric acid. A higher percentage regeneration response and shoot numbers per explant were recorded on WPM supplemented with BA and GA₃, than on MS medium containing BA and TDZ. Lower concentrations of BA and GA₃ were required for shoot elongation and prevention of shoot tip necrosis. Each cotyledonary node yielded approximately 20 shoots within 12 wk. Rooted plantlets were successfully acclimatized.     

<Emphasis Type="Italic">In vitro</Emphasis> germination and micropropagation of <Emphasis Type="Italic">Givotia rottleriformis</Emphasis> Griff.

The propagation of Givotia rottleriformis Griff. is difficult as a result of long seed dormancy associated with poor seed germination. The present study was undertaken to develop a protocol to overcome seed dormancy by culture of zygotic embryo axes and then develop an efficient method for micropropagation of Givotia. Best germination frequency (78.3%) was achieved from mature zygotic embryo axes isolated from acid-scarified fresh seeds when cultured on Murashige and Skoog (MS) medium (half-strength major salts) with 28.9 μM gibberellic acid (GA₃). Efficient plant conversion was achieved by transfer of 10-d-old germinated embryos to MS medium (half-strength major salts) supplemented with 1.2 μM kinetin (KN) and 0.5 μM indole-3-butyric acid (IBA). However, acid scarification of 1-yr-old seeds decreased the germination frequency of zygotic embryo axes in comparison to those obtained from non-acid-scarified seeds which germinated (96.2%) and converted into plants (80.3%) on MS basal (half-strength major salts) medium. Multiple shoot bud induction was achieved by culture of shoot tips derived from in vitro germinated seedlings on MS medium with 0.5 μM thidiazuron for 4 wk, and the shoots elongated after transfer to a secondary medium with 1.2 μM KN. A maximum number of 7.8 shoots per explant with an average shoot length of 3.2 cm was achieved after two subcultures on this medium. The in vitro regenerated shoots rooted (41.5%) on half-strength MS medium with 0.5 μM IBA. The in vitro generated seedlings and micropropagated plants were established in soil with a survival frequency of 70% and 60%, respectively.     

Rapid germination and development of <Emphasis Type="Italic">Taxus baccata</Emphasis> L. by <Emphasis Type="Italic">in vitro</Emphasis> embryo culture and hydroponic growth of seedlings

A highly promising procedure to obtain seedlings of Taxus baccata L. has been developed, which involves a combination of in vitro embryo culture and growth under hydroponic conditions. Embryos isolated from freshly collected seeds were 100% sterile, even though the seeds were not treated with acid or soaked in water prior to culturing. The embryo germination level of non-leached seeds was slightly lower (85%) than those leached in running water for 7 d (100%). The leached embryos germinated with extended roots while the non-leached embryos had abnormal shapes. The embryos cultured on media supplemented with an absorbent (PVP or activated charcoal) had extended roots and shoots and were a larger size without any browning, as compared to those grown without the supplement; activated charcoal gave better results. There were no significant differences in germination rates of T. baccata embryos between the media with differing strengths of macronutrients; however, for further development of the shoot, it was necessary to sub-culture the seedlings in MS in the light. To obtain seedlings with longer roots, they had to be maintained in one-half strength MS in darkness. Approximately 90% of the plants survived when grown hydroponically for 2 mo. The surviving plants showed well-extended roots and were a good starting material for genomic, proteomic, and conservational studies as well as Taxol permeabilization investigations.     

Induction and origin of adventitious roots from chimeras of <Emphasis Type="Italic">Brassica juncea</Emphasis> and <Emphasis Type="Italic">Brassica oleracea</Emphasis>

Adventitious roots were induced from shoots and leaves of the chimera plant TCC (LI-LII-LIII = TCC; T = Tuber mustard, C = Red Cabbage), previously developed by in vitro grafting of tuber mustard (Brassica juncea) and red cabbage (B. oleracea). The regeneration frequency of adventitious roots from TCC shoots and leaf sections was markedly higher than that obtained from the parents TTT (tuber mustard) and CCC (red cabbage). Moreover, levels of α-naphthaleneacetic acid in the culture medium had lower effects on rooting efficiency of TCC chimeras compared to those of TTT and CCC. The number and fresh weight of adventitious roots per TCC shoot, 13.11 roots and 0.274 g, respectively, were also significantly higher than those of the parents. This demonstrated that replacing the histogenic LI layer (the outermost apical cell layer) with a different genotype might improve adventitious root induction capability of these vegetative tissues due to likely synergistic effects between LI and the other two histogenic layers, LII and LIII. Following polymerase chain reaction analysis and histological investigation, it was found that these adventitious roots originated from the LIII histogenic layer.     

<Emphasis Type="Italic">In vitro</Emphasis> plantlet production of the endangered <Emphasis Type="Italic">Pinguicula vulgaris</Emphasis>

This study describes the development of a micropropagation protocol for Pinguicula vulgaris using cultures initiated from in vitro produced seedlings. P. vulgaris is a carnivorous plant with a northern, disjunctly circumpolar distribution and specific habitat requirements, and is hence becoming increasingly rare. Shoot proliferation was significantly influenced by Murashige and Skoog (MS) macronutrient concentration, showing higher proliferation rates in 1/4MS, but was not affected by the addition of 0.1 mg/L 6-benzyladenine (BA) or zeatin (Zea). The best medium for propagating P. vulgaris was plant growth regulator (PGR) free ¼MS. An average of 7.62 new shoots per initial explant could be obtained after 8 weeks of culture, of which over 79% produced roots during proliferation. Moreover, rooting percentages of 100% were obtained for the initial explants in all the tested media, including media without PGRs. The plantlets were successfully acclimatized to ex vitro conditions, exhibiting normal development.     

Antioxidative response of <Emphasis Type="Italic">Hordeum maritimum</Emphasis> L<Emphasis Type="Italic">.</Emphasis> to potassium deficiency

The objective of the present study was to determine the influence of potassium deprivation on the halophyte species Hordeum maritimum grown in hydroponics for 2 weeks. Treatments were with potassium (+K) or without potassium (−K). Growth, water status, mineral nutrition, parameters of oxidative stress [malondialdehyde (MDA), carbonyl groups (C=O), and hydrogen peroxide concentration (H₂O₂) contents], antioxidant enzyme activities [superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), guaiacol peroxidase (GPX, EC 1.11.1.7), ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate peroxidase (MDHAR, EC 1.6.5.4), dehydroascorbate peroxidase (DHAR, EC 1.8.5.1), and glutathione reductase (GR, EC 1.6.4.2)], and antioxidant molecules [ascorbate (ASC), and glutathione (GSH)] were determined. Results showed that the growth of vegetative organs decreased owing to potassium deficiency with roots (−36%) more affected than shoots (−12%). Water status was only diminished in roots (reduction of 24%). Potassium deprivation decreased potassium concentration in both organs, this decrease was more pronounced in roots (−81%) than in shoots (−55%). In contrast to carbonyl groups, MDA content increased owing to potassium deprivation. Except for CAT activity that remained unaffected; SOD, GPX, APX, GR, MDHAR, and DHAR activities were significantly increased. H₂O₂ concentration was negatively correlated with the activities of enzymes and the accumulation of non-enzymatic antioxidants implicated in its detoxification. In conclusion, a cooperative process between the antioxidant systems is important for the tolerance of H. maritimum to potassium deficiency.     

Endogenous polyamine profiles in different tissues of <Emphasis Type="Italic">Coffea</Emphasis> sp., and their levels during the ontogeny of fruits

Polyamines are essential compounds for growth and development in plants. An attempt has been made to find out the endogenous polyamine profiles in various parts and during the ontogeny of fruit formation of two commercially important Coffea species viz., arabica and canephora. Putrescine (Put), spermine (Spm) and spermidine (Spd) are the predominant polyamines during the ontogeny of fruit and their level increased with the advancement of fruit development. However, in the initial stages of flower and fruit development Spm levels were found to be decreased. Elevated levels of major polyamines Put, Spd, and Spm were observed in zygotic embryos than in somatic embryos. Along with this cadavarine (Cad) and other biogenic amines viz., tyramine (Tyr) and tryptamine (Try) were also found during the ontogeny of fruit in C. canephora. In this study the enodogenous polyamine profiles in coffee tissues and beans have been addressed.     

Tissue culture of <Emphasis Type="Italic">Sinningia speciosa</Emphasis> and analysis of the <Emphasis Type="Italic">in vitro</Emphasis>-generated <Emphasis Type="Italic">tricussate whorled phyllotaxis</Emphasis> (<Emphasis Type="Italic">twp</Emphasis>) variant

Two protocols were developed for the efficient regeneration of Sinningia speciosa from leaf explants via two developmental pathways. The first method involved formation of callus and buds, followed by subsequent root growth, in Murashige and Skoog medium (MS) containing 2.0 mg l⁻¹ 6-benzylaminopurine (BA) and 0.2 mg l⁻¹ α-naphthalene acetic acid (NAA), with a regeneration efficiency of 99.0%. The second method involved producing callus and roots, followed by subsequent formation of buds, in MS medium supplemented with 1.0–5.0 mg l⁻¹ NAA, and resulted in a regeneration efficiency of 90.4%. Our experiments indicate that the root-first pathway resulted in a lower plant regeneration efficiency. Through five continual generations using the buds-first method, a total of 215 regenerated plants were obtained in the last generation, and eight exhibited a phenotype we named tricussate whorled phyllotaxis (twp). Six of the regenerated twp variant plants maintained their tricussate whorled phyllotaxis phenotype, showing no other abnormalities, while one reverted to a wild type before flowering and another formed two rounds of sepals. Physiological analysis revealed that the twp plants responded differently than wild type to exogenous NAA and 2,3,5-triiodobenzoic acid (TIBA), while high-performance liquid chromatography (HPLC) analysis showed that the levels of endogenous indole-3-acetic acid (IAA) and gibberellin (GA) were lower in twp than wild-type plants. These results suggest that the formation of the twp mutant may be related to phytohormones and that the twp variant could be an important material for investigating the molecular mechanism of plant phyllotaxis patterning.