Embryo rescue and plant regeneration in banana (<Emphasis Type="Italic">Musa</Emphasis> spp.)

An efficient regeneration protocol for zygotic embryos at varying maturity stages was developed for wild banana (Pisang Jajee (AA)). Embryo ontogeny was studied to determine the best maturity stage for embryo rescue, suitable media and culture conditions (light and dark) for germination and regeneration. The conversion of endosperm from transparent fluid into a semi-solid state was followed by visible embryo development, which commenced only after 70% embryo maturity. Zygotic embryos of Pisang Jajee at different maturity levels were excised and cultured on medium fortified with different concentrations of 6-benzyl adenine (BA) and indole acetic acid (IAA). Zygotic embryos produced callus or plantlets 25 days after initiation. The frequency of callus induction was greater in immature embryos irrespective of the media composition and decreased with increasing maturity. Fully matured embryos regenerated directly into plantlets without producing callus. Immature embryos required medium supplemented with plant growth regulators (PGRs) for successful regeneration. Although the culture conditions had no influence, dark conditions favoured callus induction and plant regeneration.     

Identification of alternatively spliced <Emphasis Type="Italic">MsRan</Emphasis> transcripts involved in low temperature response in <Emphasis Type="Italic">Musa</Emphasis> spp.

Ran is involved in response to external stimuli. In this study, six MsRan gene cDNA sequences were isolated from wild banana (Musa spp. AB group) from Sanming City, China. Sequence analysis reveals that MsRan3A, MsRan3A-1a, and MsRan3C contained Ran protein domains including a GTP hydrolysis domain, a RanGAP-binding domain, and an acidic tail, whereas two G boxes (G4 and G5) were absent in MsRan3A-6a. The physicochemical property of MsRan3A, MsRan3A-1a, MsRan3A-6a, and MsRan3C appeared to differ significantly. Real time quantitative PCR (qPCR) analysis indicates that MsRan3A-1, MsRan3A-5, MsRan3A-6, MsRan3A-6a, and MsRan3C-1 were expressed in roots, leaves, peduncles, bracts, flowers, peels, and pulp of the wild banana. MsRan3A-1a was expressed at extremely low levels in these tissues and was undetectable by qPCR. The MsRan genes were found to be involved in responses to a low temperature stress but with different response patterns. Furthermore, salicylic acid significantly enhanced MsRan gene expressions suggesting the involvement of these genes in salicylic acid signal transduction.     

<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.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Acclimation of photosynthesis to temperature in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Brassica oleracea</Emphasis>

Plants differ in how much the response of net photosynthetic rate (P _N) to temperature (T) changes with the T during leaf development, and also in the biochemical basis of such changes in response. The amount of photosynthetic acclimation to T and the components of the photosynthetic system involved were compared in Arabidopsis thaliana and Brassica oleracea to determine how well A. thaliana might serve as a model organism to study the process of photosynthetic acclimation to T. Responses of single-leaf gas exchange and chlorophyll fluorescence to CO₂ concentration measured over the range of 10–35 °C for both species grown at 15, 21, and 27 °C were used to determine the T dependencies of maximum rates of carboxylation (V_Cmax), photosynthetic electron transport (J_max), triose phosphate utilization rate (TPU), and mesophyll conductance to carbon dioxide (g’_m). In A. thaliana, the optimum T of P _N at air concentrations of CO₂ was unaffected by this range of growth T, and the T dependencies of V_Cmax, J_max, and g’_m were also unaffected by growth T. There was no evidence of TPU limitation of P _N in this species over the range of measurement conditions. In contrast, the optimum T of P _N increased with growth T in B. oleracea, and the T dependencies of V_Cmax, J_max, and g’_m, as well as the T at which TPU limited P _N all varied significantly with growth T. Thus B. oleracea had much a larger capacity to acclimate photosynthetically to moderate T than did A. thaliana.     

Ultrastructural changes associated with cryopreservation of banana (<Emphasis Type="Italic">Musa</Emphasis> spp.) highly proliferating meristems

Cryopreservation has been shown to improve the frequency of virus elimination - specifically cucumber mosaic virus and banana streak virus - from banana ( Musa spp.) plants. To understand the mode of action of cryopreservation for the eradication of viral particles, we examined the ultrastructure of meristem tips at each step of the cryopreservation process. Excised meristematic clumps produced from infected banana plants belonging to cv. Williams (AAA, Cavendish subgroup) were cryopreserved through vitrification using the PVS-2 solution. We demonstrated that the cryopreservation method used only allowed survival of small areas of cells in the meristematic dome and at the base of the primordia. Cellular and subcellular changes occurring during the cryopreservation process are discussed.     

Optimization of plantain (<Emphasis Type="Italic">Musa</Emphasis> AAB) micropropagation by temporary immersion system

The positive and reliable effect of temporary immersion systems on in vitroshoot proliferation was already proved for different plant genera and it is now presented as an alternative for plantain micropropagation. Some culture parameters affecting the efficiency of the twin flasks system or temporary immersion bioreactor (Escalona et al., 1999) were investigated. Three different cytokinins (benzyladenine, thidiazuron and meta-topolin) were added to the culture medium and meta-topolin at a concentration of 4.4 M was proved to be the most efficient. Successive subcultures (28 days per subculture) were performed on medium supplemented with meta-topolin, revealing a decrease in multiplication after the 6th subculture. Multiplication rate was not changed within the ranges of immersion times (4, 12 or 22 min) and frequencies (every 3, 5 or 7 h) tested. The size of the bioreactor (250, 1,000, 5,000 or 10,000 ml) and the volume of medium per inoculum (10, 20 or 30 ml) were also evaluated and appeared to have an influence on the multiplication. A proportion of 25–100 ml of headspace per inoculum and 30 ml of medium per inoculum resulted in a multiplication rate > 13 in 28 days.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Analysis of genetic variability in two diploid <Emphasis Type="Italic">Musa</Emphasis> cultivars using RAPD

Twenty accessions of the sparsely cultivated diploid Musa cultivars Matti (AA) and Rasakadali (AB) were subjected to random amplified polymorphic DNA (RAPD) assay. A total of 14 random primers were used for the estimation of interand intracultivar variations. Out of 86 bands generated, 64 were polymorphic (74.4 % polymorphism). The cluster analysis grouped the cultivars into two major clusters: cluster I with 10 accessions of Matti and 2 of Rasakadali and cluster II comprising the remaining 8 accessions of Rasakadali. The coefficient of genetic similarity (GS) was from 0.73 to 0.99, suggesting low level of intercultivar variation. The accessions of Rasakadali with mean GS of 0.89 were genetically more diverse than those of Matti (GS = 0.93).     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Mercury Tolerance of Thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. and <Emphasis Type="Italic">Ureibacillus</Emphasis> sp

Although resistance of microorganisms to Hg(II) salts has been widely investigated and resistant strains have been reported from many eubacterial genera, there are few reports of mercuric ion resistance in extremophilic microorganisms. Moderately thermophilic mercury resistant bacteria were selected by growth at 62 °C on Luria agar containing HgCl₂. Sequence analysis of 16S rRNA genes of two isolates showed the closest matches to be with Bacillus pallidus and Ureibacillus thermosphaericus. Minimum inhibitory concentration (MIC) values for HgCl₂ were 80 μg/ml and 30 μg/ml for these isolates, respectively, compared to 10 μg/ml for B. pallidus H12 DSM3670, a mercury-sensitive control. The best-characterised mercury-resistant Bacillus strain, B. cereus RC607, had an MIC of 60 μg/ml. The new isolates had negligible mercuric reductase activity but removed Hg from the medium by the formation of a black precipitate, identified as HgS by X-ray powder diffraction analysis. No volatile H₂S was detected in the headspace of cultures in the absence or presence of Hg²⁺, and it is suggested that a new mechanism of Hg tolerance, based on the production of non-volatile thiol species, may have potential for decontamination of solutions containing Hg²⁺ without production of toxic volatile H₂S.     

Phylogeny of <Emphasis Type="Italic">Banana Streak Virus</Emphasis> Reveals Recent and Repetitive Endogenization in the Genome of Its Banana Host (<Emphasis Type="Italic">Musa</Emphasis> sp.)

Banana streak virus (BSV) is a plant dsDNA pararetrovirus (family Caulimoviridae, genus badnavirus). Although integration is not an essential step in the BSV replication cycle, the nuclear genome of banana (Musa sp.) contains BSV endogenous pararetrovirus sequences (BSV EPRVs). Some BSV EPRVs are infectious by reconstituting a functional viral genome. Recent studies revealed a large molecular diversity of episomal BSV viruses (i.e., nonintegrated) while others focused on BSV EPRV sequences only. In this study, the evolutionary history of badnavirus integration in banana was inferred from phylogenetic relationships between BSV and BSV EPRVs. The relative evolution rates and selective pressures (d_N/d_S ratio) were also compared between endogenous and episomal viral sequences. At least 27 recent independent integration events occurred after the divergence of three banana species, indicating that viral integration is a recent and frequent phenomenon. Relaxation of selective pressure on badnaviral sequences that experienced neutral evolution after integration in the plant genome was recorded. Additionally, a significant decrease (35%) in the EPRV evolution rate was observed compared to BSV, reflecting the difference in the evolution rate between episomal dsDNA viruses and plant genome. The comparison of our results with the evolution rate of the Musa genome and other reverse-transcribing viruses suggests that EPRVs play an active role in episomal BSV diversity and evolution.     

Comparative photosynthesis characteristics of <Emphasis Type="Italic">Calycanthus chinensis</Emphasis> and <Emphasis Type="Italic">Chimonanthus praecox</Emphasis>

Calycanthus chinensis is an endangered plant of the national second-grade protection of China restricted in a small area in Zhejiang Province. We studied parameters of photosynthesis, chlorophyll (Chl) contents, and Chl fluorescence (minimum fluorescence, F₀, maximum fluorescence, F_m, variable fluorescence, F_v, and F_v/F_m) of C. chinensis and Chimonanthus praecox. C. chinensis had lower compensation irradiance but higher saturation irradiance than C. praecox. Hence C. chinensis has more advantage in obtaining and utilizing photon energy and higher Chl content, and is more adaptive to higher temperature and propitious to thermal dissipation than C. praecox. In addition, C. chinensis produces abundant, well-preserved seed with a higher germination rate and a wider adaptability to temperature than C. praecox. Thus C. chinensis is prone to survival and viability, and gets rid of the endangered plant species of the national second-grade protection of China.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.     

Activities of photosystem II and antioxidant enzymes in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) cultivars exposed to chilling temperatures

This study was carried out to determine the effect of chilling on both cold-acclimated and non-acclimated chickpea (Cicer arietinum L.) cultivars (Gökçe and Can?tez 87). Chickpea seedlings grown in soil culture for 12 days were subjected to chilling temperatures (2 and 4°C for 12 days) after maintaining in cold-acclimation (10°C, 7 days) or non-acclimation (25°C, 7 days) periods. The lowest values of growth parameters were obtained with cold-acclimated plants, whereas non-acclimated plants exhibited the lowest water content values, especially at 2°C. There was no effect of cold-acclimation period on chlorophyll fluorescence parameters. Plants subjected to chilling temperatures after cold-acclimation were more tolerant with respect to chlorophyll fluorescence parameters, and Gökçe had better photosystem II (PSII) photochemical activity. In the chilling treatments, total chlorophyll (a + b) content reduced, especially at 2°C, while anthocyanin and flavonoid contents increased to a greater extent in Gökçe and carotenoid content of the cultivars did not change. Malondialdehyde (MDA) content was higher for Can?tez 87, mostly at 2°C, while proline accumulation was greater for Gökçe. The cold-acclimation period led to a remarkable increase in antioxidant enzyme activities of both cultivars. The superoxide dismutase (SOD) activity was much higher in Gökçe for both chilling temperatures and the ascorbate peroxidase (APX) activity increased only in the cold-acclimated 4°C treatments. Similarly, with APX activity, the glutathione reductase (GR) and peroxidase (POD) activities of cultivars were higher in cold-acclimated plants at both the chilling temperatures, mostly in Gökçe. The results of this study indicate that cold-acclimation increased the cultivars ability to withstand the chilling temperatures. The lower MDA content and higher antioxidant and photochemical activities in Gökçe indicated an enhanced chilling tolerance capacity of this cultivar to protect the plant from oxidative damage.     

Isolation and <Emphasis Type="Italic">S</Emphasis>-genotyping application of <Emphasis Type="Italic">S</Emphasis>-allelic polymorphic <Emphasis Type="Italic">MdSLFB</Emphasis>s in apple (<Emphasis Type="Italic">Malus domestica</Emphasis> Borkh.)

Apple (Malus domestica Borkh.) possesses gametophytic self-incompatibility (GSI) which is controlled by S-RNase in the pistil as well as a pollen S-determinant that has not been well characterized. The identification of S-locus F-box brother (SFBB) genes, which are good candidates for the pollen S-determinant in apple and pear, indicated the presence of multiple S-allelic polymorphic F-box genes at the S-locus. In apple, two SFBB gene groups have been described, while there are at least three groups in pear. In this report, we identified five MdSLFB (S-RNase-linked F-box) genes from four different S-genotypes of apple. These genes showed pollen- and S-allele-specific expression with a high polymorphism among S-alleles. The phylogenetic tree suggested that some of them belong to SFBBα or β groups as described previously, while others appear to be different from SFBBs. In particular, the presence of MdSLFB3 and MdSLFB9 suggested that there are more S-allelic polymorphic F-box gene groups in the S-locus besides α and β. Based on the sequence polymorphism of MdSLFBs, we developed an S-genotyping system for apple cultivars. In addition, we isolated twelve MdSLFB-like genes, which showed pollen-specific expression without S-allelic polymorphism.     

High-level overproduction of <Emphasis Type="Italic">Thermus</Emphasis> enzymes in <Emphasis Type="Italic">Streptomyces lividans</Emphasis>

Biotechnology needs to explore the capacity of different organisms to overproduce proteins of interest at low cost. In this paper, we show that Streptomyces lividans is a suitable host for the expression of Thermus thermophilus genes and report the overproduction of the corresponding proteins. This capacity was corroborated after cloning the genes corresponding to an alkaline phosphatase (a periplasmic enzyme in T. thermophilus) and that corresponding to a beta-glycosidase (an intracellular enzyme) in Escherichia coli and in S. lividans. Comparison of the production in both hosts revealed that the expression of active protein achieved in S. lividans was much higher than in E. coli, especially in the case of the periplasmic enzyme. In fact, the native signal peptide of the T. thermophilus phosphatase was functional in S. lividans, being processed at the same peptide bond in both organisms, allowing the overproduction and secretion of this protein to the S. lividans culture supernatant. As in E. coli, the thermostability of the expressed proteins allowed a huge purification factor upon thermal denaturation and precipitation of the host proteins. We conclude that S. lividans is a very efficient and industry-friendly host for the expression of thermophilic proteins from Thermus spp.