<Emphasis Type="Italic">In vitro</Emphasis> regeneration,flower and plant formation from petiolar and nodal explants of culantro (<Emphasis Type="Italic">Eryngium foetidum</Emphasis> L.)

Summary A procedure for plant regeneration, flower and plant formation from petiolar and inflorescence nodal explants of culantro is discribed. Leaf petioles were excised from young leaves of non-flowering plants while nodal explants were excised from the inflorescence. Explants were cultured in Murashige and Skoog (MS) medium alone or supplemented with 0.5μM naphthaleneacetic acid (NAA) and 0.9, 1.8, 4.5, or 9 μM thidiazuron (TDZ). All explants produced multiple shoots. In addition, nodal explants formed flowers. Shoot number, flower number and shoot length were influenced by TDZ and NAA. Rooted shoots from both types of explants were transferred to soil where plants were successfully established.     

An improved micropropagation protocol for teak

An improved micropropagation protocol has been developed for teak (Tectona grandis). Nodal explants placed on MS medium supplemented with 22.2 M benzylaminopurine and then serially transferred to fresh medium after 12, 24, 48 and 72 h gave maximum culture establishment (76.8%). Establishment was reduced when explants were retained in the initial culture medium longer than 12 h. Explants collected in May showed maximum (76.8%) response. Placement of the explants on MS medium supplemented with 22.2 M benzylaminopurine and 0.57 M indole-3-acetic acid resulted in the maximum average number of shoots. In vitro raised micro shoots were rooted ex vitro by dipping in indole-3-butyric acid (9.8 mM) for 2 min followed by planting in polyethylene pots containing a soil:vermiculite (1:1 v/v) mixture. This treatment resulted in 77.9% survival of the plantlets. They were weaned in a glasshouse and finally moved to an agro-net shade house.     

An improved protocol for<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated transformation of<Emphasis Type="Italic"> Antirrhinum majus</Emphasis> L.

Efficient Agrobacterium -mediated transformation of Antirrhinum majus L. was achieved via indirect shoot organogenesis from hypocotyl explants of seedlings. Stable transformants were obtained by inoculating explants with A. tumefaciens strain GV2260 harboring the binary vector pBIGFP121, which contains the neomycin phosphotransferase gene (NPT II) as a selectable marker and the gene for the Green Fluorescent Protein (GFP) as a visual marker. Putative transformants were identified by selection for kanamycin resistance and by examining the shoots using fluorescence microscopy. PCR and Southern analyses confirmed integration of the GFP gene into the genomes of the transformants. The transformants had a morphologically normal phenotype. The transgene was shown to be inherited in a Mendelian manner. This improved method requires only a small number of seeds for explant preparation, and three changes of medium; the overall transformation efficiency achieved, based on the recovery of transformed plants after 4–5 months of culture, reached 8–9%. This success rate makes the protocol very useful for producing transgenic A. majus plants.Communicated by G. Jürgens     

Influence of growth regulators on plant regeneration from epicotyl and hypocotyl cultures of two groundnut (Arachis hypogaea L.) cultivars

This study was designed to evaluate the effect of phytohormones on plant regeneration from epicotyl and hypocotyl explants of two groundnut (Arachis hypogaea) cultivars. Explants cultured on media with auxins and in combination with cytokinin produced high frequency of callus. After four weeks, callus from these cultures was transferred to medium with cytokinin and reduced auxin, shoot buds regenerated from the cultures. A high rate of shoot bud regeneration was observed on medium supplemented with 2.0 mg/L BAP and 0.5 mg/L NAA. Among the different auxins tested, NAA was found to be most effective, producing the highest frequency of shoot buds per responding cultures. Of the two explants tested, epicotyl was found to be best for high frequency shoot bud regeneration. Multiple shoots arose on MS medium supplemented with BAP or kinetin (1.0–5.0 mg/L) plus IBA (1.0 mg/L), with maximum production occurring at 5.0 mg/L. The elongated shoots developed rootsin vitro upon transfer to MS medium supplemented with NAA or IBA (0.5–2.0 mg/L) and kinetin (0.5 mg/L) for 15 days.In vitro produced plantlets, were transferred to soil and placed in a glasshouse developed successfully, matured, and set seeds.     

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.     

Roles for the N- and C-terminal domains of phytochrome B in interactions between phytochrome B and cryptochrome signaling cascades

Plants fine-tune light responses through interactions betweenphotoreceptors. We have previously reported that the greeningof Arabidopsis thaliana roots is regulated synergistically byphytochromes and cryptochromes. In the present study, we investigatedthe functions of the N- and C-terminal domains of phytochromeB (phyB) in the interactions between phyB and cryptochrome signalingcascades. Transgenic Arabidopsis expressing the phyB N-terminaldomain fused to green fluorescent protein (GFP), ß-glucuronidase(GUS) and the nuclear localization signal (NLS) showed intenseroot greening under blue light, indicating that the C-terminaldomain was dispensable for the synergistic interaction in theinduction of root greening. However, root greening under redlight was substantially reduced in the absence of the C-terminaldomain. This effect was opposite to the previous observationthat removal of the C-terminal domain enhanced the signalingactivity of phyB in the inhibition of hypocotyl elongation.In addition, we found that overexpression of the isolated C-terminaldomain of phyB enhanced the blue light response not only forroot greening but also for the inhibition of hypocotyl elongation.Analysis of this activity on various photoreceptor mutant backgroundsdemonstrated that the isolated C-terminal domain enhanced cryptochromesignaling. In summary, these results demonstrate that differentdomains of phyB can play various roles which are dependent onlight conditions as well as on the specific physiological response.     

Effect of high oxygen on placental function in short-term explant cultures

Ex situ culture of human gestational tissues has been routinely used as a model to investigate tissue function. The objective of this study was to determine the effect of varying oxygen concentrations on human term placental explants over a 24-h time period. Specifically, the effect of incubating placental explants in oxygen concentrations of 8%, 21% or 95% on tissue viability, metabolism and cell death was measured by assessing glucose consumption, lactate production, release of lactate dehydrogenase, parathyroid hormone-related protein (PTHrP), tumour necrosis factor-alpha (TNF-α) and 8-isoprostane, immunoreactivity for cleaved-caspase-9 and immunohistochemistry for the caspase-3-cleaved cytokeratin-18 neoepitope, M30. Exposure to higher oxygen concentrations significantly increased the rates of glucose consumption and lactate production. Apoptosis was significantly increased under conditions of higher oxygen as evidenced by increased M30 in placental explant sections. Similarly, hyperoxia significantly increased the releases of PTHrP, TNF-α and 8-isoprostane. Thus, incubation of placental explants with oxygen concentrations of 95% and, to a lesser extent, 21% oxygen was associated with the modulation of multiple cellular response pathways including those associated with tissue viability and cell death. These data are consistent with the hypothesis that hyperoxia activates pathways and mechanisms involved in cellular metabolism, necrosis and apoptosis, thereby shifting the balance from a steady state towards cell death.     

Direct shoot regeneration from nodal explants of <Emphasis Type="Italic">Sida cordifolia</Emphasis> Linn

A method was developed to initiate multiple shoots from mature nodal explants of Sida cordifolia Linn. High frequency of regeneration was achieved on Murashige and Skoog (MS) medium supplemented with 2.0 mg l⁻¹ 6-benzylaminopurine, 0.5 mg l⁻¹ α-naphthalene acidic acid, 1.0 mg l⁻¹ adenine sulfate, and 10% (v/v) coconut milk. Multiple shoots were initiated within 21 d and the above media was capable of inducing the formation of more than 20 shoots from each explant. Regenerated shoots were successfully rooted on half-strength MS medium supplemented with 2.0 mg l⁻¹ indole-3-butyric acid and 3% (w/v) sucrose. Rooted plantlets were established in soil. The regenerated plantlets showed no morphological differences from the parent material. This protocol could be useful for germplasm conservation, cultivation, and genetic improvement of S. cordifolia.     

TSA1 interacts with CSN1/CSN and may be functionally involved in Arabidopsis seedling development in darkness

The COP9 signalosome (CSN) is a multiprotein complex which participates in diverse cellular and developmental processes.CSN1,one of the subunits of CSN,is essential for assembly of the multiprotein complex via PCI (proteasome,COP9 signalosome and initiation factor 3) domain in the C-terminal half of CSN 1.However,the role of the N-terminal domain (NTD) of CSN 1,which is critical for the function of CSN,is not completely understood.Using a yeast two-hybrid (Y2H) screen,we found that the NTD of CSN1 interacts with TSK-associating protein 1 (TSA1),a reported Ca2+-binding protein.The interaction between CSN1 and TSA1 was confirmed by co-immunoprecipitation in Arabidopsis.tsal mutants exhibited a short hypocotyl phenotype in darkness but were similar to wild-type Arabidopsis under white light,which suggested that TSA1 might regulate Arabidopsis hypocotyl development in the dark.Furthermore,the expression of TSA1 was significantly lower in a csnl null mutant (fus6),while CSN1 expression did not change in a tsal mutant with weak TSA1 expression.Together,these findings suggest a functional relationship between TSA1 and CSN1 in seedling development.     

Cytokinin-induced hypocotyl elongation in light-grown<Emphasis Type="Italic"> Arabidopsis</Emphasis> plants with inhibited ethylene action or indole-3-acetic acid transport

Cytokinins inhibit hypocotyl elongation in darkness but have no obvious effect on hypocotyl length in the light. However, we found that cytokinins do promote hypocotyl elongation in the light when ethylene action is blocked. A 50% increase in Arabidopsis thaliana (L.) Heynh. hypocotyl length was observed in response to N⁶-benzyladenine (BA) treatment in the presence of Ag⁺. The level of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid was strongly increased, indicating that ethylene biosynthesis was up-regulated by treatment with cytokinin. Furthermore, the effects of cytokinins on hypocotyl elongation were also tested using a series of mutants in the cascade of the ethylene-signal pathway. In the ethylene-insensitive mutants etr1-3 and ein2-1, cytokinin treatment resulted in hypocotyl lengths comparable to those of wild-type seedlings treated with both Ag⁺ and BA. A similar phenotypical response to cytokinin was observed when auxin transport was blocked by -naphthylphthalamic acid (NPA). Applied cytokinin largely restored cell elongation in the basal and middle parts of the hypocotyls of NPA-treated seedlings and at the same time abolished the NPA-induced decrease in indole-3-acetic acid levels. Our data support the hypothesis that, in the light, cytokinins interact with the ethylene-signalling pathway and conditionally up-regulate ethylene and auxin synthesis.